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