Introduction To Molecular Biology (2) PDF - 2022-2023

Introduction to molecular biology (2) Pr. Raouf Alami CM1 Module : cell biology and molecular biology...

Introduction to molecular biology (2) Pr. Raouf Alami CM1 Module : cell biology and molecular biology Molecular biology Année Universitaire: 2022-2023 www.um6ss.ma Course contents I. Ribonucleic acid (RNA) RNA and DNA RNA size RNA backbone RNA structures RNA functions RNA integrity (Methods to check) Types of RNA mRNA Non-coding RNAs Transfert RNA (tRNA) Ribosomal ribonucleic acid (rRNA) RNA length II. Organization of eukaryotic chromosomes The nucleosome Chromatin higher-order structure Linker histones Euchromatin versus heterochromatin Chromosomes Karyotype Telomeres III. Human genome Human genome project Genetic polymorphism Quiz 2 Ribonucleic acid (RNA) RNA is a linear polymer Ribonucleic acid is a molecule that is present in the majority of living organisms and viruses. It is made up of nucleotides, which are ribose sugars attached to nitrogenous bases and phosphate groups. The nitrogenous bases include : adenine, guanine, uracil, and cytosine RNA is similar to DNA structure but involved in different cellular functions Riboncleotides Similarly, for orientation purpose the Ribose sugar is numbered from 1 to 5, with coresponding 1’and 5’ ends Similar to the DNA structure. RNA conists of « basic elements: a sugar, a phosphate group and 4 nitrogenous bases Adjoining nucleotides add on to the 3’ hydroxyl group to the previous nucleotide  3 major differences with DNA 1- RNA is a single strand molecule , wich is more instable than DNA : Allows flxibility to bend back on itself allowing weak bonds with other parts of the same molecule RNA has 3 major differences with DNA 2- It has a ribose sugar instead of a deoxyribose in the DNA wich is important for enzyme that distinguish DNA from RNA RNA has 3 major differences with DNA 3- RNA has a Uracil nitrogene base instead of thymine allowing base pair complementary rule Adenine binds to Uracil Cytosine binds to Guanine  DNA/RNA CH3 8 RNA size Most RNAs are no more than a few thousand nucleotides long, and many are considerably shorter. A DNA molecule in a human chromosome can be up to 250 million nucleotide-pairs long; According to the length of RNA chain, RNA includes small RNA and long RNA. Usually, small RNAs are shorter than 200 nt in length. Large RNAs are greater than 200 nt long. (could be coding RNA (=mRNA) or non coding RNAs) RNA backbone RNA structures Three-dimensional arrangement of RNA, include helical duplexes and other components  RNA functions RNA performs many functions in an organism, such as coding, decoding, regulating, and expressing genes. About 5% of the weight of a human cell is RNA. Only about 1% of a cell consists of DNA. RNA is found in both the nucleus and cytoplasm of humans cells RNA Functions : mRNA carries genetic codes from the DNA in the nucleus to ribosomes, for protein translation with the help of tRNA and rRNA Other types of RNAs primarily carry out biochemical reactions, similar to enzymes. Other types have complex regulatory functions in cells. Such as microRNA, coding (cRNA) and noncoding RNA (ncRNA) Methods to Check RNA Integrity The most common method used to assess the integrity of total RNA is to run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). Agarose Gels The 28S rRNA band should be rRNAs are found in the ribosomes and account approximately twice as for 80% of the total RNA present in the cell. intense as the 18S rRNA band  Types of RNA mRNA, rRNA, and tRNA are the three main types of RNA. Which RNA is maximum amount in a cell? r-RNA counts to about 80% of the total RNA present in the cell. It is present in ribosomes. t-RNA counts to about 15% of the total RNA. mRNA accounts for about 5% of the total RNA in the cell  Messenger RNA First described in 1961 by Jacob and Monod coined the name "messenger RNA". Messenger RNA (mRNA) is a copy of DNA. It’s the coding RNA (cRNA) mRNA carries information from DNA to the ribosome, the sites of protein synthesis (translation) in the cell. mRNA carries a “message” from the nucleus to the cytoplasm. Example of mRNA size: Human α- and β-globin mRNAs are 575 and 626 nucleotides in length  Transfert RNA tRNA is the smallest of the 3 types of RNA, possessing around 75-95 nucleotides. tRNAs are an essential component of translation, where their main function is the transfer of amino acids during protein synthesis.  Ribosomal ribonucleic acid (rRNA) 1. Ribosomal ribonucleic acid (rRNA) is a non-coding RNA 2. It is the primary component of ribosomes. 3. Ribosomal RNA is bound to ribosomal proteins to form small and large ribosome subunits. In the ribosomes of humans, The Small Subunits (SSU) contains a single small rRNA. The large subunit (LSU) contains one large RNA, one small RNAS, and about 35 different proteins, most of them in single copy. Non-coding rNAS About 97% of the transcriptional output is non-protein-coding in eukaryotes Non-coding RNAs ("ncRNA") can be encoded by their own genes (RNA genes). Example the rRNA, tRNA. ❑ There are also non-coding RNAs involved in Gene regulation, RNA processing and other roles. (Certain RNAs are able to catalyze chemical reactions such as cutting and ligating other RNA molecules) Non-coding RNAs are arbitrarily classified into short and long non-coding RNA based on transcript size  Example of a non coding RNA The hammerhead ribozyme is used for targeted RNA cleavage experiments 19 Example of a non coding RNA These results demonstrate that it is possible to decrease the concentration of βS chains and mRNA with the help of a hammerhead ribozyme. 20 Organization of Eukaryotic chromosomes - Human genome - 21 Organization of Eukaryotic chromosomes Chromatin was first discovered by using dyes to stain it. Walther Flemming (21 April 1843 – 4 August 1905) In 1974, it was first proposed by Roger Kornberg that chromatin was based on a repeating unit of a histone octamer and around 200 base pairs of DNA Organization of Eukaryotic chromosomes A long DNA molecule with a diameter of 2 nm is wrapped around a core histone with a diameter to form an 11 nm fiber The nucleosome are then folded into 30 nm ~ 147 bp chromatin fibers. The 30 nm fiber is compacted into the chromatin with a diameter of about 0.7 µm. Eukaryotes nucleus size : around 5-10 μm  The nucléosome The nucleosome is an octamer proteins histones namely : H2A, H2B, H3 and H4. Solenoid A solenoid is a condensed chromatin fiber with 30nm diameter. It helps to package eukaryotic DNA into the nucleus. Solenoid = helical form 1400nm  Chromatin higher-order structure Chromatin is the complex of genomic DNA with proteins called histones, where each histone-bound DNA molecule is referred to as a chromosome. Chromatin higher-order structure is any assemblage of nucleosomes in 3D space. The most obvious chromatin higher-order structure is the mitotic/meiotic chromosome in which the DNA is compacted some 10,000- to 20,000-fold  Linker histones (H1) Histone 1 (H1) = linker histone. The linker histone H1 binds to the entry/exit sites of DNA on the surface of the nucleosomal core particle and completes the nucleosome. It is required to stabilize the 30-nm fibre (or higher- order chromatin structures) In mammals, there are at least eight H1 subtypes, including H1s, H1a to H1e,H1o, … They could have many gene expression consequences 27  Linker histones (H1) importance of linker histone To determine whether the H1 histone subtypes play differential roles in the regulation of gene expression, we combined mice lacking specific H1 histone subtypes with mice carrying transgenes subject to position effects. We report that some, but not all, linker histones can attenuate or accentuate position effects. The results suggest that the linker- histone subtypes play differential roles in the control of gene expression and that the sequential arrangement of the linker histones on the chromatin fiber might regulate higher-order chromatin structure and fine-tune expression levels. Euchromatin versus heterochromatin Similarities between euchromatin and heterochromatin There are so many differences between the two types of chromatins, though, they have some similarities as well. 1. Both are sequences of DNA made up of polynucleotide chains. 2. The euchromatin and heterochromatin are visible more distinctly during the interphase stage of the cell division. The real image of banded chromosomes shows 3. Both chromatins can interact with the euchromatin region and heterochromatin region on chromosome histones to package on chromosomes. Differeces between euchromatin and heterochromatin Euchromatin has a less compact structure, whereas heterochromatin is more compact and composed of an array of nucleosomes condensed into a fiber. Euchromatin is a form of chromatin that is enriched in genes, and is often under active transcription Heterochromatin is defined as the area of the chromosome which is darkly stained with a DNA specific stain and is in comparatively condensed form. Differeces between euchromatin and heterochromatin Euchromatin versus heterochromatin  Chromosomes Chromosomes are composed of condensed chromatin fibers. Chromosomes are the highest level of organization of DNA and proteins. The main function of chromosomes is to carry the DNA and transfer the genetic information from parents to offspring. Chromosomes play an important role during cell division. They protect the DNA from getting tangled and damaged. Karyotype A karyotype is an individual’s complete set of chromosomes. The term refers to a laboratory-produced image of a person’s chromosomes isolated from an individual cell and arranged in numerical order. A karyotype may be used to look for abnormalities in chromosome number or structure. The typical human karyotype contains 22 pairs of autosomal chromosomes and one pair of sex chromosomes. The most common karyotypes for a female contain In normal diploid organisms, autosomal chromosomes are two X chromosomes and are denoted for the sex XX. present in two copies. Males usually have both an X and a Y chromosome, denoted for the sex XY.  Telomeres TTAGGG. 1. Telomeres are sections of DNA, found at the ends of each of our chromosomes. 2. They consist of the same sequence of bases repeated over and over. 3. In humans the telomere Illustration showing the position of telomeres at the end of our chromosomes. Image credit: Genome Research Limited sequence is TTAGGG. 4. This sequence is usually repeated about 3,000 times and can reach up to 15,000 base pairs in length.  Telomeres 1. They allow the chromosome to be replicated properly during cell division: Every time a cell carries out DNA replication the chromosomes are shortened by about 25-200 bases per replication. 2. However, because the ends are protected by telomeres, the only part of the chromosome that is lost, is the telomere, and the DNA is left undamaged. 3. Without telomeres, important DNA would be lost every time a cell divides (usually about 50 to 70 times). 4. This would eventually lead to the loss of entire genes. Centromere The centromere links a pair of sister chromatids together during cell division. This constricted region of chromosome connects the sister chromatids, creating a short arm (p) and a long arm (q) on the chromatids. During mitosis, spindle fibers attach to the centromere via the kinetochore. Centromeres play essential roles in equal chromosome segregation by directing the assembly of the microtubule Centromeres typically are in silent or gene-free chromosome regions but may include genes and are commonly transcribed at low levels to form non-coding RNAs The human genome The human genome is a complete set of nucleic acid sequences for humans, encoded as DNA within the 23 chromosome pairs in cell nuclei and in a small DNA molecule found within individual mitochondria. Human genomes include both protein-coding DNA sequences and various types of DNA that does not encode proteins.  Human genome The nuclear genome comprises approximately 3 200 000 000 nucleotides of DNA, divided into 46 DNA linear molecules, the shortest 50 000 000 nucleotides in length the longest 260 000 000 nucleotides, each contained in a different chromosome. An international research effort called the Human Genome Project, which worked to determine the sequence of the human genome and identify the genes that it contains, estimated that humans have between 20,000 and 25,000 genes. Every person has two copies of each gene, one inherited from each parent. It represents about 2% of the whole genome In genetics, the term junk DNA refers to regions of DNA that are noncoding. DNA contains instructions (coding) that are used to create proteins in the cell.  Human Genome Project Sequencing Human Genome: the Contributions of Francis Collins and Craig Venter The public Human Genome Project (HGP), which was launched in 1990, was to sequence the 3 billion bases of the human genome. This announcement led to cooperation between Collins and Venter, and on June 26, 2000, Venter and Collins jointly announced that, after nearly a decade of work, both the public Human Genome Project Dr. Francis Collins and J. Craig Venter joined President Bill Clinton headed by Collins and Celera Genomics headed by Venter had at the White House on Monday to announce the completion of the deciphered essentially all the genes in human DNA. first draft of the human genome. June 26th, 2000 Craig Venter, founder of the Institute for Genomic Research in Maryland, USA The gold standard sequence was released in 2003  Genetic polymorphism Polymorphism, as related to genomics, refers to the presence of two or more variant forms of a specific DNA sequence that can occur among different individuals or populations. The most common type of polymorphism involves variation at a single nucleotide (also called a single-nucleotide polymorphism, or SNP). Other polymorphisms can be much larger, involving longer stretches of DNA. Single-nucleotide polymorphism (SNP)  Genetic polymorphism Genetic polymorphism A tandem repeat is a sequence of two or more DNA bases that is repeated numerous times in a head-to-tail manner on a chromosome. Tandem repeats are generally present in non-coding DNA. In some cases, tandem repeats can serve as genetic markers to track inheritance in families. They can also be useful for DNA fingerprinting in forensic studies. Short tandem repeats (strs) As individuals will likely have different numbers of repeats at a given satellite DNA locus, they will generate unique DNA profiles Comparative STR Lengths at Two Specific Loci Genetic polymorphism Forensic Investigations: Suspects should be a complete match with the DNA sample taken from the crime scene if a conviction is to occur. The number of loci used to generate a unique profile depends on the size of the population being compared : E.g. ❑America (population: ~ 320 million) uses 13 loci for comparison; ❑Australia (population: ~ 25 million) uses only 9 loci Genetic polymorphism Example: Use of Forensic tandemInvestigation repeats in Compare the DNA collected from the crime scene to determine which of the investigations three suspects was present (click picture to solve) Genetic polymorphism Genetic polymorphism Paternity Testing: Children inherit half their chromosomes from each parent and thus should possess a combination of parental fragments In other words, all fragments produced in the child should also be produced by either the mother or father Paternal Testing Compare the profiles of three men with that of a mother and child to determine the biological father (click picture to solve)  Quiz Quiz How many chromatin make up a chromosome? There is one chromatin per one chromosome. How many DNA molecule make up a chromosome? There is one DNA molecule per one chromosome.  Quiz Name 3 major differences between RNA and DNA 1. RNA is a single strand molecule 2. RNA has a ribose sugar 3. RNA has a Uracil What makes Rna backbone? Phosphate and Ribose 47  Quiz What is he most common method used to assess the integrity of total RNA To run an aliquot of the RNA sample on a denaturing agarose gel and look for rRNA When mRNA was first described? described in 1961 by Jacob and Monod What role tRNA plays in a cytoplasm? Essential component of translation 48  Quiz Give an example of a non coding RNA tRNA, rRNA RNA can play an important role as enzymes, can name a type of this kind of RNA? hammerhead ribozyme When Chromatin was first described 1880 by Flemming How many base pair are wrapped around a nucleosome? About 147 bp How many proteins make a nucleosome? 8 How many times the DNA needs to be compacted to form a chromosome? 10,000- to 20,000-fold 49  Quiz What are the H1 histones Linker histones (entry/exit of DNA from two consecutive nucleosomes) What are the similarities between euchromatine and heterochromatine? made up of nucleotides are visible more distinctly during the interphase stage of the cell division. can interact with histones to package on chromosomes 50  Quiz What are the differeces between euchromatin and heterochromatin Euchromatin has a less compact structure, whereas heterochromatin is more compact Heterochromatin is darkly stained with a DNA specific stain and is in comparatively condensed form. Which form is found in prokaryotes not in eukaryotes? Euchromatine Which one is the last to replicate ? Heterochromatine The human genome is composed of how much heterochromatine? About 98% What the most important role of chromosomes? They protect the DNA from getting tangled and damaged. 51  Quiz What is a karyotype? The term refers to a laboratory-produced image of a person’s chromosomes isolated from an individual cell and arranged in numerical order. What the human sequence of the telomere? TTAGGG (TT a GGG) This sequence is repeated many time it could reach :? can reach up to 15,000 base pairs in length What would happened if there is no telomeres on a chromosome? Without telomeres, important DNA would be lost every time a cell divides (usually about 50 to 70 times). 52  The centromere links a pair of sister Quiz What the role of centromeres? chromatids together during cell division. How many DNA molecule in a human saumatic cell.? 46 DNA linear molecules, What junk DNA means? In genetics, the term junk DNA refers to regions of DNA that are noncoding. When was the human gold standard sequence released? 2003 What SNP means in genetics? Single-nucleotide polymorphism How can we use the tandem repeat sequence ? Tandem repeats can serve as genetic markers to track inheritance in families. They can also be useful for DNA fingerprinting in forensic 53 studies