Basics in Molecular Biology - UniFI - DiSIA · Basics in Molecular Biology Gerolamo Lanfranchi...
Transcript of Basics in Molecular Biology - UniFI - DiSIA · Basics in Molecular Biology Gerolamo Lanfranchi...
Basics in Molecular Biology
Gerolamo Lanfranchi
Department of Biology and CRIBI Biotechnology Centre
Università degli Stdi di Padova, Italy
Functional Genomics: Principles of Statistics and Biology
Centro Studium San Domenico
May 9-13, 2005 Florence
The cell is the fundamental unit of living organisms
According to the number of these units, organisms can be divided in:
Unicellular organisms (bacteria, fungi, protozoa)Multicellular organisms
Cells have very different morphologies and functions, that could change during development, differentiation and aging (neuron, muscle, erythrocyte)
Description of the canonical structure of model cells:Prokaryotic cellEukaryotic cell
Section of a typical bacterial cell
CapsulaeOuter membrane
Cell wallinner membrane
Nucleoid region(DNA)
Ribosomes
Pili
Flagellum
Eukaryotic cells
The cell can be regarded as a self-replicating unit that contains in itself all the information needed for:
- building and maintenance of its structural components- basic and differentiated function (e.g.: respiratory metabolism,
contraction, production of proteins, signal transmission, etc.)- transmission of these information to daughter cells through the ordered
process of cell division (mitosis) or to a novel organism (meiosis)- functional interaction and cooperation with other cells of the organism- changes of structural components and function during growth,
differentiation and aging (cell or organism)- changes of structural components and functions in response to internal
or external stimuli (other cells, infectious agents, molecules such as hormones, physico/chemical stimuli , environmental stimuli)
- cell senescence and death- cell transformation and carcinogenicity
The information is contained in the DNAVery long molecules (chromosomes, genome)
Section of a bacterial cell
CapsulaeOuter membrane
Cell wallinner membrane
Nucleoid region(DNA)
Ribosomes
Pili
Flagellum
DNA CHEMISTRY
DNA (deoxyribonucleic acid, sodium salt) is made by long molecules composed by a linear series of units called
nucleotides
Each nucleotide is composed by three chemical units:
- phosphate
- sugar
- base
Bases and therefore nucleotides, can be of four types:
DNA - Adenine (A), Thymidine (T), Cytidine (C) and Guanine
Ribonucleic acid (RNA)
RNA - Adenine (A), Uracyl (U), Cytidine (C) and Guanine
DNA is a complex but ordered molecule
A single chain of DNA is made by joining different nucleotides through a chemical bond between the phosphate group of a nucleotide and the sugar of the next nucleotide
Each chain of DNA has is unique sequence of bases or nucleotides. The sequence can be read as:
5’ – T A C G – 3’
Chargaff rule:
A – T (A – U)
G – C
THE DOUBLE HELIX
(Watson – Crick)DNA molecules in the chromosomes are made by two antiparallel, self-complementary chains of nucleotides
The two chains (strands) are kept together by hydrogen bonds between opposite bases
Very long DNA molecules are tightly packed into the chromosomes
Chromosomes are confined in the nucleus
Chromosomes are visible only during cell division
This chromosome is composed by two double-helix DNA molecules that have an identical sequence of bases
All cells of a multicellular organism have the same number of chromosomes. The number and structure of chromosomes is stable
THE CELL DIVISION (MITOSIS) - 1The DNA content is stable during the cell life-cycle, is duplicated before cell division and one complete genome is fully inherited by each daughter cell after cell division
The DNA content (genome) is stably transmitted to progeny cells
THE CELL DIVISION (MITOSIS) - 2The DNA content is stable during the cell life-cycle, duplicated before cell division and one complete genome is fully inherited by each daughter cell after cell division
The DNA content (genome) is stably transmitted to progeny cells
DNA REPLICATION
At the molecular level in the chromosomes, before cell division, in each DNA molecule the two antiparallel chains are first separated and a novel chain is synthesized on the old one by a precise enzymatic machinery
Remember the Chargaff rule
At the end of DNA replication two identical DNA molecules are generated which will move in the two daughter cells
Information of the genome of the mother cell is therefore conserved in the daughter cells (same nucleotide sequence)
The genomic DNA contains all the information for the structure, function evolution and behavior of cells and the whole organism (the genetic program).
All cells of a multicellular organism contain the same genome (amount and sequence)
The genetic program is determined mostly by genes
Genes are distinctive fragment of DNA dispersed in the genome
Genomes have very different gene content in their genomic DNA
Some genomes are very crowded by genes
e.g.: phage lambda, 90% of genomic DNA
In some genomes genes are highly dispersed
e.g.: in human genome genes are only 2.5%, 97,5% junk DNA
The genomic DNA contains all the information for the structure, function evolution and behaviour of cells and the whole organism (the genetic program).
All cells of a multicellular organism contain the same genome (amount and sequence)
The genetic program is determined mostly by genes
Genes are distinctive fragment of DNA dispersed in the genome
Some genomes are filled by genes
- phage lambda, 90% of genomic DNA are genes
In some genomes genes are highly dispersed
- in the human genome genes are only 2.5%, 97,5% junk DNA
The information enclosed in the genes should flow from the nucleus
GENE EXPRESSION
RNA synthesis (TRANSCRIPTION) and transport in the cytoplasm
Differences between prokaryotes and eukaryotes
RNA transcription is performed by enzymatic complexes (RNA polymerases) that copy specific strand of genomic DNA (genes)
into a complementary single-stranded molecule of RNA
Transcription of RNA starts at the beginning of a gene and stops at its end
Initiation signals (Promoters) and stop signals (terminators)
The result of this reaction is a messenger RNA molecule identical to the gene
The messenger RNA is a single-stranded nucleic acid molecule
Only one strand of a gene is copied by the RNA polymerase
Number of genes:
E. coli: 4,289
Yeast: 6,300
Drosophila: 14,000
Arabidopsis: 26,000
Human: 38,000 (?)
Not all the genes of a genome are active in a given cell
Cell morphology, phisiology and function are so different because different genes are switched ON an OFF
REGULATION OF GENE EXPRESSION
In human muscle cell 15,000/38,000 genes are expressed, in brain cell 30,000/38,000
The regulation of gene expression acts at different levels: the first is regulation of transcription
REGULATION OF TRANSCRIPTION 1 (level):
within a cell:
some genes are highly transcribed (many mRNA molecules)
some genes are weakly transcribed (few mRNA molecules)
Notes: the level of transcription is not related to the “relevance” of a given gene in the cell; e.g. transcription factors that regulate the expression of other genes are normally weakly transcribed
The less abundant transcripts are very difficult to measure
B B B B BB B B B BB B B B BB B B B BB B B B B
A
REGULATION OF TRANSCRIPTION 2 (tissue-specificity):
The same genes can be differentially transcribed in different cells, tissues, organs of a multicellular organism
Cell, Tissue, Organ N. 1 Cell, Tissue, Organ N. 2
REGULATION OF TRANSCRIPTION 3 (splicing):
Genes are organized in EXONS (coding fragments = translatable intoprotein) and INTRONS (non-coding fragments)
mRNAs are maturated and modified before translation into proteins
REGULATION OF TRANSCRIPTION 3 (alternative splicing):
mRNAs can be maturated and modified in differet ways before translation
Different ways of joining EXONS
REGULATION OF TRANSCRIPTION 3 (alternative splicing):
Differently maturated mRNAs form the same gene are expressed in different cells or tissues
REGULATION OF TRANSCRIPTION 4 (during development):
Genes with the same function have been duplicated during evolution and slightly differentiated (gene families)
Different gene members of a gene family can be transcriptionally switched on and off in different stages of organism development
REGULATION OF TRANSCRIPTION 5 (environmental):
Genes respond to external signals varying their level of transcription
Specific signal molecules can enter the nuclei, bind to gene promoters and activate, repress or modulate the transcription of specific genes
The regulation of gene transcription is mediated by specific set of proteins (transcription factors and co-factors that bind to the regulatory region of a gene (promoters, enhancers, LCRs), modulating the RNA Polymerase activity on that specific gene
The structure of the promoter regions of an eukaryotic gene and regulatory proteins binding to them
Phenotypic and functional differences between different tissues of a multicellular organism are indeed due to differential transcription activity of hundreds of genes
Gene 1
Tissuesample 1
Red = over expressed genes
Green = down regulated genes
Black = not detectable
Genes can be isolated, sequenced and studied by the use of theircorresponding messenger RNA (mRNA)
Total RNA can be purified from cells, tissues, organs, organisms
Different types of RNAs in the cell:
- rRNA (80%)
- small RNAs (15%)
- mRNA (2-5%) derived by gene transcription
Different types of RNA in an eukaryotic cell
TOTAL RNA
mRNAs have a long tail of A at their end
mRNAs can be purified from the rest of total RNA through specific binding of poly-A tails to short stretches of complementary T bound to solid surfaces.
The whole content of polyadenilatedmRNAs (transcribed genes) of a cell, tissue, organism, can be purified in this way
THE TRANSCRIPTOME
mRNAs are very unstable molecules and single-stranded
To be cloned, the mRNAs should be transformed into complementary molecules of double-stranded DNA
The cDNA
mRNAs are converted into cDNAs by a series of enzymatic reactions carried out completely in vitro.
The sequence of nucleotides of each original mRNA is exactly maintained in the corresponding cDNA (stable information)
Cloning of cDNAs into plasmid vectors (self-replicating DNA molecules that can be hosted and maintained in bacteria)
cDNA library
Thousands of cDNAs
cDNAs insertedinto plasmids
plasmidsintroduced intobacteria
Preparing a cDNA library
Collection of bacterial clones
Each clone contains and replicates a specific plasmid with the sequence corresponding to a single cDNA (gene)
Two different approaches for studying gene transcription:
a) Measuring single transcripts (genes)
- a cloned cDNA corresponding to the mRNA to be measured (PROBE)
- mRNA population purified from the target tissue, cells (TARGET)
b) Measuring the entire set of mRNAs of a cell, tissue (the transcriptome)
Probe for a single mRNA (gene)
mRNAs from tissueA, B, C 1 - Measuring a single transcript
mRNA RECOGNITION BY NUCLEIC ACID HYBRIDIZATION
Single-stranded labeled probe (from a cloned cDNA) that recognizes a specific target mRNA bound to filters
A A C
PERFECT MATCH
PARTIAL MATCH
Tissue A Tissue B
cDNA library2 - Measuringthe
transcriptome