Regulation of Gene Expression

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Regulation of Gene Expression in Eukaryotes:

Regulation of Gene Expression in Eukaryotes Dinesh D. Khedkar Shri Shivaji Science College, Amravati

Regulation of Gene Expression:

Regulation of Gene Expression Bacterial Gene Regulation Single chromosome with very small size One cistron Short Life span Eukaryotic Gene Regulation Many chromosome with Huge Genome Polycistronic Long life span

Regulatory steps:

Transcription Translation Protein Targeting / Sorting Protein Trafficking Protein Functioning Regulatory steps

Regulatory steps:

Regulatory steps

FINE STRUCTURE OF GENE:

FINE STRUCTURE OF GENE The hereditary units which are transmitted from one generation to the next generation arc called genes. A gene is the fundamental biologic unit Mendel first of all conceived of the genes as particulate units and referred as hereditary factors or hereditary elements. Wilhelm Johansen coined the term gene in 1909 to describe heritable factors responsible for the transmission and expression, but without reference to any particular theory of inheritance Chromosomal Theory of Inheritance

NATURE OF GENE:

NATURE OF GENE De Vries one gene one character hypothesis Bateson and Punnett the presence or absence theory . But all the theories were discarded by Morgan, who produced the particulate gene theory in 1926 . He considered genes as corpuscles which are arranged in a linear order on the chromosomes and appear like beads on a string. But, the discovery of DNA molecule as a sole carrier of genetic information has altogether discarded the Morgan's theory. Therefore, before defining the gene it will be advisable consider the both classical as well as modern definitions of gene.

CLASSICAL GENE:

CLASSICAL GENE Classically, gene as assumed to be a genetic unit by the following three criteria: ( i ) A gene is a unit of physiological function that occupies a definite locus in the chromosome and is responsible for a specific phenotypic character, (ii) A gene is a unit of transmission or segregation , because it can be segregated and exchanged at meiosis by way of crossing over. (iii) A gene is a unit of mutation , because by a spontaneous or induced change it can give rise to different phenotypic expression. These classical definitions of genes have been revised thoroughly due to recent discoveries in the field of molecular genetics .

CLASSICAL GENE:

CLASSICAL GENE This concept was known as classical gene structure. It states that Genes determine physical as well as physiological characteristics. Genes are present on the chromosome, and on a single chromosome there are many genes Genes occupy a specific position on a chromosome which is called as locus or loci and genes are arranged in single linear order

MODERN GENE – Benzer’s Concept:

MODERN GENE – Benzer’s Concept Modern Definition of Gene After the discovery of DNA, its parallel behaviour with that of chromosomes and proper understanding of most of the molecular phenomena which may interplay in the determination of a phenotypic trait, the gene has been defined as follows:

CISTRON:

CISTRON The portion of DNA specifying a single polypeptide chain is termed as cistron , which is a synonym for the termed, the gene of physiological function. The term cistron has been coined by Seymour Benzer . Haemoglobin , therefore, would require two cistrons for its globin protein fraction, one each for the α and β chains. A cistron for α -chain has at least 141 X 3-423 nucleotides and the citron for the β-chain 146 X 3=438 nucleotides.

MUTON:

MUTON There are many positions or sites within a cistron where mutations can occur. Therefore, the gene as a unit of mutation is smaller. i.e., it consists of fewer nucleotides than a cistron . Benzer coined the word muton to that smallest length of DNA capable of mutational change. Thus, a muton can be defined as the smallest unit of genetic material which when changed or mutated produces a phenotypic effect. A muton may thus be delimited to a single nucleotide or some part of nucleotide. Different forms of a mutationality defined genes are called homoalleles . For example, in bacteria muton may be nucleotide pair and in cistron for haemoglobin the muton may be single nucleotid .

RECON:

RECON Sometimes crossing over or recombination occurs in a cistron and this provides still, other sub-divisional concept of the cistron , namely the recon. A recon is the smallest unit of DNA capable of recombination or of being integrated by transformation in bacteria. Recombinationally separable forms of a cistron are called heteroalleles

MODERN GENE:

All geneticists are not in agreement with these definitions of gene, however following definition of gene may serve our purpose. "The gene of function is the sequence of nucleotides (providing numerous sites for intragenic mutation and or/recombination) which specifies the amino acid sequence of a specific polypeptide chain." MODERN GENE

PowerPoint Presentation:

What is Gene ? A gene is a segment of nucleic acid that contains the information necessary to produce a functional product, usually a protein. Genes consist of a long strand of DNA (RNA in some viruses) that contains Regulatory Sequences (a promoter, which controls the activity of a gene, and a coding sequence, which determines what the gene produces). A T G C

PowerPoint Presentation:

What is Gene ? A gene is a segment of nucleic acid that contains the information necessary to produce a functional product, usually a protein . Genes consist of a long strand of DNA (RNA in some viruses) that contains Regulatory Sequences (a promoter, which controls the activity of a gene, and a coding sequence, which determines what the gene produces). A T G C

PowerPoint Presentation:

What is Gene ? Genes are poly-nucleotides. Each nucleotide is made up of a base group, a sugar and one phosphate group The sugar group forms the backbone of DNA. The phosphate group are responsible for linking one nucleotide to another . The nitrogenous bases of nucleotides face each other and form Hydrogen bonds with their complimentary bases.

Guinness Book of protein-coding genes (Human):

Guinness Book of protein-coding genes ( Human )

Guinness Book of protein-coding genes (bacteria):

Guinness Book of protein-coding genes (bacteria)

Operons – the basic concept of Prokaryotic Gene Regulation:

Operons – the basic concept of Prokaryotic Gene Regulation Regulated genes can be switched on and off depending on the cell’s metabolic needs Operon-a regulated cluster of adjacent structural genes, operator site, promotor site, and regulatory gene(s)

Operon:

Operon Structural gene -gene that codes for a polypeptide Promoter region -controls access to the structural genes, located between the promoter and structural genes, contains the operator site. Operator Site -region where the repressor attaches Regulatory genes -codes for repressor proteins Polycistronic mRNA -transcript for several polypeptides

Lac Operon:

Lac Operon Regulator Promoter Operator Lac Z Lac Y Lac A BPs +/- 111 - 35 -26 0 3063 800 800 Peptide Amino acid 360 1021 275 275 MW ( Da ) 3800 1,25,000 30,000 30,000 Active Protein Tetramer Tetramer Monomer Dimer Function Repressor β - Galactosidase Permease Trans acetylase

Repressible vs. Inducible Operons two types of negative gene regulation:

Repressible vs. Inducible Operons two types of negative gene regulation Repressible Operons Genes are initially ON Anabolic pathways End product switches off its own production Inducible Operons Genes are initially OFF Catabolic pathways Switched on by nutrient that the pathway uses

lac: an inducible operon:

lac : an inducible operon

An example of positive gene regulation-cAMP:

An example of positive gene regulation- cAMP cAMP exerts positive control Binds to promoter, stimulating transcription Dependent on glucose concentration

Eukaryotic gene organization:

Eukaryotic gene organization enhancers silencers

The Control of Gene Expression:

The Control of Gene Expression Only a few genes are active at any time-differential gene expression Control can be exerted at any step in the pathway. Chromatin modifications affect availability of genes for transcription

Transcriptional regulation via Chromatin modification:

Transcriptional regulation via Chromatin modification DNA methylation -methyl groups added to cytosine-inactivate genes Histone acetylation - -COCH 3 added to amino acids. Reduce binding between DNA and histone -consequence? TRANSCRIPTION INITITION

Control at the transcriptional level:

Control at the transcriptional level Transcription Factors -augment transcription by binding to DNA or to each other. Act as repressors and activators. Coordinately controlled genes -usually associated with a specific regulatory sequence and activated or repressed by the corresponding transcription factor

PowerPoint Presentation:

Opportunities for the control of gene expression in the eukaryotic cell

Transcription Initiation:

Transcription Initiation Diversity of RNA Polymerase RNA Pol Location Product I Nucleolus rRNA II Nucleoplasm Nuclear RNA III Nucleoplasm T & small RNA

RNA Polymerase – I :

RNA Polymerase – I Two part promoter Core Promoter - -45 to + 20 Upstream control Element - -110 to -170 UCE (Upstream Control Elem) and UBP TBP (Tata Binding Factor)

RNA Polymerase – III :

RNA Polymerase – III Upstream Control Element and Downstream Control Element Promoters – UCE – TATA, PSE, OCT DCE – Box A, Box B and Box C (for Transcription Factors attachment)

RNA Polymerase – II :

RNA Polymerase – II Can not initiate itself completely depend on TF Enzyme with TF constitute Basal Transcription Appratus Most of the promoter has TATA, CAAT & GC Box Upstream Control Element and

PowerPoint Presentation:

-140 -120 -80 -60 -40 -20 Start Point CAAT GC TATA

TATA BOX:

TATA BOX -25 bp Upstream of the start point 8 bp Consensus sequence entirely of AT Tend to be surrounded of GC rich region Single base mutation acts as a strong down mutation RNA Pol – II initiate at this point TATA Binding Protein (TBP) (30,000 Da ) and TBP Associated Factors (TAF) forms appratus Interacting with other TFs

TATA BOX:

TATA BOX TBP is a commitment factor working for positioning RNA Pol. Binding in Wide Groove Mutation in TATA Box leads to erratic initiation Bound to ~ 10 bp

PowerPoint Presentation:

1. TFIID : multiprotein complex including TBP, other proteins are known as TAF II s TBP is the only protein binds to TATA box

PowerPoint Presentation:

3. TFIIB & RNA Pol binding binds to TFIID Binds to RNA Pol with TFIIF

CAAT BOX:

CAAT BOX Located at -75 to – 80 Upstream Consensus Sequence Functions in either orientation Bound to ~ 22 bp Its inclusion leads to increase promoter strength CTF and NF-1 are the assisting factors 3 Lacs /cell

GC BOX:

GC BOX Located at -90 Upstream Consensus Sequence of GGGCGG Multiple copies, Functions in either orientation Bound to ~ 20 bp Its inclusion leads to increase promoter strength SP-1 are the assisting factors 60,000 /cell

PowerPoint Presentation:

Example: The metallothionein (MT,) gene The metallothionein protein protects the cell against excess concentrations of heavy metals , by binding the metal and removing it from the cell. The gene is expressed at a basal level, but is induced to greater levels of expression by heavy metal ions (such as cadmium) or by glucocorticoids.

The Structure of Chromatin:

The Structure of Chromatin DNA complexed with protein forms chromatin diffuse during interphase condensed during mitosis, forms chromosomes histones and nucleosomes

The structure of Chromatin:

The structure of Chromatin Based on successive levels of DNA packing

The structure of Chromatin (2):

The structure of Chromatin (2) Six nucleosomes/turn, forms a cylinder Higher level of DNA packing: looped domains (20,000 to 100,000 nucleotides ) Heterochromatin remains highly condensed during interphase (Barr bodies) Euchromatin able to be transcribed during interphase

Types of Chromatin:

Types of Chromatin Heterochromatin : Highly condensed during interphase, not actively transcribed Euchromatin : Less condensed during interphase, able to be transcribed

PowerPoint Presentation:

Regulation of gene expression at the level of chromatin Sequence-independent linker histones: control DNA compaction and accessibility to trans -acting factors post-translational modifications of histone tails: control compaction of DNA and serve as docking sites for trans-acting factors Range: Can act at the level of a single gene, often acts over groups of genes and over larger domains (20-200kb), and can affect gene expression over an entire chromosome

The Code Beyond Genetics in DNA:

The Code Beyond Genetics in DNA The original code is that each codon specifies a particular amino acid and subsequent protein The second code is determined by the placement of the nucleosomes. Nucleosomes protect and control access to the DNA

Nucleosomes:

Nucleosomes 30,000,000 nucleosomes in each human cell DNA wraps 1.65 times around a nucleosome The DNA twist is 147 base pairs The average DNA strand contains 225 million base pairs Made of proteins called histones

How do Nucleosomes Function?:

How do Nucleosomes Function? Bind to the DNA at specific sequences Prevent transcription factors from attaching and initiating transcription Nucleosomes can and do move, letting DNA open to be transcribed. How? This has not yet been determined !

The Genetic Basis of Development:

The Genetic Basis of Development From single cell to multicellular organism Differential gene expression Genetic and cellular mechanisms of pattern formation

From single cell to multicellular organism:

From single cell to multicellular organism Involves cell division, morphogenestis and cell differentiation Cell division : increases cell numbers Morphogenesis : overall shape of the organism is established Cell differentiation : cells become specialized in structure and function Development has been studied using model organisms

Differential Gene Expression:

Differential Gene Expression Different types of cells in an organism have the same DNA Plants are totipotent , cells retain the ability of the zygote to give rise to all differentiated cells Animals are not as plastic, alternative approaches used, nuclear transplantations such as “Dolly”

Determination:

Determination Different cell types make different proteins Role of transcription regulation Two sources of cellular instructions for determination: cytoplasmic determinants and neighboring cells

Genetic and Cellular Mechanism of Pattern Formation:

Genetic and Cellular Mechanism of Pattern Formation Pattern Formation: spatial organization of tissues and organs characteristic of the mature organism Plants-continuous process throughout life Animals-restricted to embryos and juveniles

Homologous genes that affect pattern formation:

Homologous genes that affect pattern formation

How genes control development (Genetic analysis of Drosophila):

How genes control development (Genetic analysis of Drosophila ) Revealed roles of specific molecules that direct position and differentiation Cytoplasmic determinants provide postional information (unfertilized eggs: orientation of anterior-posterior and dorsal-ventral already determined) 1200 genes essential for development, 120 in segmentation

Role of Gradients of Maternal Molecules:

Role of Gradients of Maternal Molecules Hypothesized over 100 years ago Bicoid Gene essential for development of the anterior of a fly, produces mRNA that concentrates in anterior half of unfertilized eggs. Female flies w/out this gene produce embryos lacking front half of embryo Bicoid protein regulate other genes, a domino like effect

Homeotic Genes: What are they?:

Homeotic Genes: What are they? Master regulatory genes that identify specific regions of the body and appropriate placement of appendages contain a sequence of 180 nucleotides called the homeobox identical or similar homeobox sequences have been identified in many other invertebrates, vertebrates, fungi and prokaryotes.

What is a homeobox?:

What is a homeobox ? "Since their discovery in 1983, homeobox genes, and the proteins they encode, the homeodomain proteins, have turned out to play important roles in the developmental processes of many multicellular organisms. While certainly not the only developmental control genes, they have been shown to play crucial roles from the earliest steps in embryogenesis

PowerPoint Presentation:

The homeobox was originally described as a conserved DNA motif of about 180 base pairs. The protein domain encoded by the homeobox , the homeodomain , is thus about 60 amino acids long. The first genes found to encode homeodomain proteins were Drosophila developmental control genes, in particular homeotic genes, from which the name " homeo"box was derived. However , many homeobox genes are not homeotic genes; the homeobox is a sequence motif, while " homeotic " is a functional description for genes that cause homeotic transformations."

summary:

summary Thus, one can say that the homeodomain is a DNA-binding domain that occurs in proteins that are usually transcription factors. These transcription factors regulate the transcription of other genes and hence very frequently play important roles in development of multicellular organisms.

Some definitions:

Some definitions Hox genes : Hox genes are a subgroup of homeobox genes. In vertebrates these genes are found in gene clusters on the chromosomes. In mammals four such clusters exist, called Hox clusters. The gene name " Hox " has been restricted to name Hox cluster genes in vertebrates. Only genes in the HOX cluster should be named Hox genes. So note: homeobox genes are NOT Hox genes, Hox genes are a subset of homeobox genes.

HOX cluster::

HOX cluster : The term Hox cluster refers to a group of clustered homeobox genes, named Hox genes in vertebrates, that play important roles in pattern formation along the anterior-posterior body axis. In fact, the first homeobox genes discovered where those of the Drosophila homeotic gene clusters, i.e. the " Antennapedia complex" and the " Bithorax complex", which summarily are referred to as HOM-C ( homeotic complex). This HOM-C complex in Drosophila is the evolutionary homolog of the vertebrate Hox clusters and the evolutionarily related homeobox gene clusters in other animals (i.e. chordates, insects, nematodes, etc.) are now also called HOX clusters.

PowerPoint Presentation:

homeodomain : a DNA-binding domain, usually about 60 amino acids in length, encoded by the homeobox . homeobox : a fragment of DNA of about 180 basepairs (not counting introns ), found in homeobox genes.

Role of Neighboring Cells-Induction:

Role of Neighboring Cells-Induction Signaling help coordinate spatial and temporal expression of genes sequential inductions control organ formation results in selective activation and inactivation of genes within target cells

Apoptosis-programmed cell death:

Apoptosis-programmed cell death “suicide” genes- product present continuously depends upon regulating protein activity tadpole tail? Degenerative diseases, cancers-faulty apoptotic mechanisms?

The Molecular Biology of Cancer:

The Molecular Biology of Cancer Genetic changes that affect the cell cycle (viruses, carcinogens) Oncogene -cancer-causing gene Proto-oncogenes - normally code for regulatory proteins controlling cell growth, division, and adhesion

The Molecular Biology of Cancer:

The Molecular Biology of Cancer Result of genetic changes -can be random -can be caused by viruses or carcinogens Oncogenes: cancer causing genes -formed from proto-oncogenes by DNA movement within the genome; gene amplification, or point mutations changes in tumor-suppressor genes

Proto-oncogenesOncogenes:

Proto-oncogenes Oncogenes Movement of DNA within the genome Gene amplification Point mutation Sometimes suppressor genes that normally inhibit growth can be responsible for cancer

Multiple mutations underlie the development of cancer:

Multiple mutations underlie the development of cancer 15% due to viruses Somatic mutations ( 5-10% of breast cancer)

Posttranscriptional Mechanisms:

Posttranscriptional Mechanisms Regulation of mRNA degradation : several hours or even weeks Protein processing and degradation : activation may require addition of phosphate groups or sugars; use of signal sequences; marking for destruction Control of translation : inactivation of initiation factors, use of repressor proteins

Posttranscriptional Mechanisms:

Posttranscriptional Mechanisms May be stopped or enhanced at any posttranscriptional step Role of the nuclear envelope Regulation of mRNA degradation- several hours to several weeks Control of translation- inactivation of initiation factors, use of repressor proteins Protein processing and degradation- may require addition of sugars or phosphates; use of signal sequences; marking for destruction

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