Fundamentals of Molecular Biology 2024 PDF

PLBL 21532 Fundamentals of Molecular Biology Prepared by Dr. Harshini Herath Department of Plant and Molecular Biology 2024 Conducted by Dr. Vihara Kaluthanthri 1 Gene Expression Regulation Eukaryotes Multicellul...

PLBL 21532 Fundamentals of Molecular Biology Prepared by Dr. Harshini Herath Department of Plant and Molecular Biology 2024 Conducted by Dr. Vihara Kaluthanthri 1 Gene Expression Regulation Eukaryotes Multicellular organism, ▪ All cells contain the same genome. ▪ But different cells types express different sets of genes. ▪ Structure and function of different cell types differ significantly  Constitutive genes (Housekeeping genes) ▪ Expressed at constant levels regardless of cell environmental conditions ▪ Products are required to maintain basic cellular structure and functions; ▪ DNA and RNA polymerases ▪ Actin ▪ GAPDH ▪ Ubiquitin ▪ rRNA ▪ tRNA ▪ Ribosomal proteins ▪ Enzymes catalyzing housekeeping functions  Non-constitutive genes ▪ Expressed only when the gene products are required ▪ Under certain environmental conditions ▪ E.g. Hormones ▪ Regulated  Steps at which gene expression can be regulated Chromatin accessibility For most genes, ▪ Transcription initiation is the most important point of control Ensures that cells will not synthesize excessive intermediates Gene expression regulation steps - Bacteria vs Eukaryotes ▪ Repressors ▪ Activators ▪ DNA modification Transcriptional regulation ▪ Histone modification ▪ Epigenetic gene regulation ▪ Regulatory RNA Epigenetics  Genetics ▪ The study of genes and heredity (inheritance) Epigenetics ▪ The study of heritable changes in gene function that do not involve changes in the DNA sequence. epi (Greek word) Over/Outside of In addition to traditional genetic basis of inheritance ▪ Transmission of non genetic information (epigenetic states) from an organism to its offspring. ▪ The transmission of gene expression patterns (epigenetic inheritance) ▪ Two individuals with same DNA sequence at a locus show different phenotypes. Mechanisms that produce such phenotypic changes: 1. DNA methylation 2. Histone modifications 3. Higher-order chromatin structures 4. Noncoding RNA, Antisense RNA and RNA Interference ▪ Change gene expression without changing DNA sequence ▪ Gene expression regulation in eukaryotes 1. DNA methylation ▪ CG islands ▪ Regions with a high frequency of CG ▪ CpG islands ▪ Cytosines can be methylated to form 5-methylcytosine ▪ By DNA methyltransferases (Dnmt) ▪ Mostly at promoters 2. Histone modifications ▪ Histone proteins in chromatin : H2A, H2B, H3, H4 ▪ Histone methylation ▪ Promote heterochromatin formation ▪ Silencing modifications ▪ Histone 3 lysine 9 (H3K9) ▪ Histone 4 lysine 20 (H4K20) ▪ Histone 3 lysine 27 (H3K27) 3. Higher-order chromatin structures ▪ Heterochromatin formation ▪ By ▪ Heterochromatic protein 1 (HP1) ▪ Polycomb group proteins 4. Noncoding RNA, Antisense RNA and RNA Interference ▪ Gene silencing mechanisms ▪ Regulate genomic imprinting X chromosome inactivation Mammals ▪ Females XX chromosomes ▪ Males XY chromosomes ▪ X chromosome Large More than 1000 genes ▪ Y chromosome Smaller Less than 100 genes ▪ Female cells contain two copies of X chromosome genes as do male cells. Female mammalian embryo ▪ Early development ▪ One X chromosome in each cell becomes highly condensed into heterochromatin ▪ X-inactivation ▪ Barr body ▪ Located near nuclear membrane ▪ Two X chromosomes differ in their expression Dosage compensation mechanism ▪ To equalize the dosage of X chromosome gene products between males and females ▪ To maintain the correct ratio of X chromosome gene products to autosome gene products ▪ Achieved by ▪ Transcriptional inactivation of one X chromosome in female somatic cells ▪ X-inactivation Which X chromosome is inactivated? ▪ Random ▪ Maternally inherited chromosome (Xm) ▪ Paternally inherited chromosome (Xp) How is an entire X chromosome transcriptionally inactivated? ▪ Inactivation initiates from the middle of X chromosome ▪ X-inactivation center (XIC) ▪ DNA sequence: Approx. 106 nucleotide pairs ▪ Regulatory element ▪ Starts heterochromatin formation ▪ Facilitates bi-directional spread of heterochromatin formation along entire X chromosome Mammalian X-chromosome inactivation Many features of mammalian X-chromosome inactivation remain to be discovered. XIC ▪ Encodes an RNA ▪ XIST RNA (X-inactivation specific transcript) ▪ Not translated to a protein ▪ Necessary for X-inactivation ▪ Remains in nucleus ▪ Coats inactive X chromosome ▪ Form and spread heterochromatin X-chromosome heterochromatin ▪ Contains ▪ XIST RNA ▪ A specific variant of histone 2A ▪ Hypoacetylated histones H3 and H4 ▪ Histone H3 methylated at a specific position ▪ Methylated DNA ▪ Making inactive X chromosome resistant to transcription When is the X-chromosome inactivated? ▪ After several thousand cells have formed in the embryo ▪ Female has groups of cells in which either Xp or Xm is silenced. ▪ Distributed in small clusters in adult animal Clonal inheritance of a condensed inactive X chromosome in female mammals E.g. Some female cats ▪ Red and black coat coloration (tortoise-shell) ▪ One X chromosome: A gene for red hair color ▪ Other X chromosome: Allele of same gene for black hair color ▪ Random X-inactivation - Produces patches of cells of 2 distinctive colors ▪ Male cats ▪ Solid red or solid black ▪ Depending on which X chromosome they inherit from mother Once an X-chromosome is inactivated, ▪ Maintained silent over many cell divisions ▪ During many cycles of DNA replication and mitosis ▪ Epigenetic inheritance: Phenotype does not depend on the DNA sequence X-chromosome inactivation is not always permanent. ▪ Reversed during germ cell formation ▪ Haploid oocytes contain an active X chromosome ▪ Express X-linked gene products  Genomic imprinting The regulation of genes whose expression depends on whether they are maternally or paternally inherited. Imprint ▪ A mark (outline) on a surface Imprinted gene ▪ An epigenetically silenced gene In diploid organisms, ▪ Two alleles of a gene are inherited − one from mother and one from father. ▪ For most genes, both alleles are expressed. ▪ For some genes only one allele is expressed, while the other allele is inactive. ▪ As a result of imprinting, that occurs through marking of a gene by epigenetic mechanisms during gamete production ▪ Important for normal development of the organism Genomic imprinting ▪ An epigenetic process that marks DNA in a sex dependent manner, resulting in differential expression of a gene depending on its parent of origin. ▪ Stable male and female imprints are established during gametogenesis ▪ Imprinted states are maintained through DNA replication in somatic cells of embryo ▪ Imprinted states are maintained throughout the life of organism ▪ Preceding generation’s imprint is erased in germ line and a new imprint is re-established according to the sex of the organism E.g. Gene for insulin-like growth factor-2 (Igf2) ▪ An imprinted gene ▪ Required for prenatal growth of mice ▪ Only the paternal copy is transcribed ▪ Paternal gene mutated Stunted mice ▪ Maternal gene defective Normal mice Imprinting mechanisms 1. DNA methylation 2. Histone modifications 3. Higher-order chromatin structures 4. Noncoding RNA, Antisense RNA and RNA Interference ▪ Change gene expression without changing DNA sequence  Epigenome The collection of all of the epigenetic marks on the DNA in a single cell Epigenome vs Cancer Epigenomic map ▪ A diagrammatic representation of gene expression, DNA methylation and histone modification status of a particular genomic region. Techniques used to study epigenetics ▪ Chromatin immunoprecipitation ▪ Fluorescent in situ hybridization ▪ Methylation-sensitive restriction enzymes ▪ DNA adenine methyltransferase identification ▪ Bisulfite sequencing ▪ Bioinformatics (computational epigenetics)