introduction to molecular biology and basic terminology

FACULTY OF TROPICAL AGRISCIENCES

CZECH UNIVERSITY OF LIFE SCIENCES PRAGUEPlant breeding and genetic resources conservation

Basic terminology &

Introduction to molecular biology



Basic terminologyGenome– complete set of genes in an organismChromosomes– the nucleoprotein structures that carry the genetic informationGene– the determinant of an observable trait or characteristic of an organism

(pigmentation)– the DNA sequence that determines the chemical structure of a specific

polypeptide molecule or RNA moleculeAllele– a viable DNA coding that occupies a given locus (position) on achromosome– specific form of a gene (blue flower…)



Basic terminologyHistone– highly alkaline proteins found in eukaryotic cell nuclei that order the DNA molecules into structural units – nucleosome– condensation of DNANucleosome– basic repeating structural and functional unit of chromatin– contains nine histone proteins and about 166 base pairs (bp) of DNAChromatine– resulting DNA-protein complex



Gene structureExon – coding regionIntron – non-coding regionBoth are transcribed into pre-mRNA, but only exons are translated into proteins



DNA RNA

Deoxyribonucleic acid2’-deoxy-D-riboseAdenineGuanine CytosineThymine

Ribonucleic acidD-riboseAdenineGuanine CytosineUracil

-Monomer units of nucleic acids are nucleotides (sugar + base + phosphate)

-Each nucleotide is a phosphate ester of a corresponding nucleoside (sugar and base)

-Base order is called sequence



DNADiscovered in 1953 by James Watson and Francis Crick(Nobel prize in Physiology or Medicine 1962 together with Maurice Wilkins)



DNA- Two polynucleotide chains (primary

structure) forming a double helix (secondary structure)

- Prevails right-handed helix (B-DNA), but other conformations exist

- Conformation depends on:Ions and hydratationDNA sequencePresence of special proteins

- DNA is compacted more than a thousandfold

- Antiparallel strands



DNA structure



DNA – base pairing- Watson-Crick pairing- Purines bound to pyrimidines- Bound by hydrogen bonds

(H-bonds), which are quite weak

- A•T (two bonds)- C•G (three bonds)- Sugars bound together by

phosphodiester backbone



Types of DNA

According to the number of the strands:- dsDNA- ssDNA

According to the shape:Circular

- Covalently Closed Circle (CCC Dna)

- Open circle (OC Dna)LinearAccording to the location in the cell:

Nuclear (nDNA)Non-nuclear

- Mitochondrial (mtDNA)- Chloroplast (cpDNA)- Plasmide



Enzymes influencing DNA

Deoxyribonuclease I (Dnase I) DNA ligaseBinds to the minor grooveNonspecifically cleaves DNA molecule to small piecesReleases di-, tri- and oligonucleotide products

Joins Okazaki fragments during replicationCompletes short-patch DNA synthesis occurring in DNA repair process



Enzymes influencing DNARestriction endonucleases

Obtained from bacteria (protection against viral infections – non-methylated sequences)Cleaves both DNA strands in highly specific pointsRecognize palindrome(base sequence that reads the same in one direction on one strand as it does the other direction on the other strand)Create blunt or sticky ends



TopoisomerasesOne of the components of chromatinInterconvert different topological states of DNAChange the level of supercoilingTopoisomerase I cleaves one strandTopoisomerase II cleaves both strands



RNAMultiple roles in cellsCatalyzators (protein synthesis, splicing…)Scaffold subcellular structuresRegulation of gene expressionKey component for the protein systhesis



Types of RNAMessenger RNA (mRNA) Transfer RNA (tRNA)

Conveys sequence information from the genome to the protein synthetic apparatusConsists of codons(Sequence of three adjacent nucleotides constituting the genetic code that specifies the insertion of an amino acid in a specific structural position in a polypeptide chain during the synthesis of proteins)

Synthesized complementary and antiparallel to the template strand

Brings activated molecules of amino acids to ribosomesContains anticodon2 functions:

Activation (by esterification) of amino acidsAdaptor molecules (for every AA there is at least one tRNA)



Types of RNARibosomal RNA (rRNA)

Decodes mRNAs into amino acids (fabricates polypeptides)2 subunits: the large (LSU, 60S) and the small one (SSU, 40S)SSU protein, initiation factors and the initiator tRNA, binds to AUG in the 5' region of the mRNA



DNA replicationEnsures the transmission of genetic information to daughter cellsEvery new cell contains one strand from the mother cell and one new-synthesized strandNecessity of a primer - a short sequence of RNA from which DNA replication can initiateOriC – initiation of the replicationCarries out in 5’-3 direction, but on both strandsInteraction of many enzymesOkazaki fragment – short fragment of DNA synthesized on lagging strand



Replication – enzymesDNA helicase – separation of two strandsDNA single-stranded binding proteins – stabilisation of the single strand created by helicaseDNA gyrase – catalyses the formation of negative supercoilPrimase – synthetises RNA primer

DNA Polymerase α –5' to 3' DNA-dependent DNA polymerase 5' to 3' DNA-dependent RNA polymerase (primase)

DNA Pol. δ –3' to 5' exonuclease (proof-reading activity) 5' to 3' DNA polymerase

DNA Pol. ε –5' to 3' DNA-dependent DNA polymerase3‘ to 5' exonuclease activity

Ligase – binds the lagging strand



Replication

Video available on URL: http://www.youtube.com/watch?v=teV62zrm2P0



Transcribing of genetic information from DNA to RNACatalyzed by RNA polymerase II (RNAP II)DNA contains many genes lined up one after another

TranscriptionmRNA contains the message for one gene (eukaryotes), or for several adjacent genes(prokaryotes)Starts at promoter (TATA box) –binding site for RNAP and transcription factors



TranscriptionDNA is unwound:

Sense strand (untranscribed)Antisense strand (3’-5’; transcribed)

Carries on in 5’-3’directionEnds at terminator



TranscriptionWhole gene ranscribed to so-called pre-mRNA (precursor mRNA)Methyl guanine (“cap”) added at the beginning of the pre-mRNANon-coding sequences (introns) have to be cut off

Activity of small nuclear ribonucleoproteins (snRNPs) –intron forms a loop and is cut offRemaining exons joined together – RNA splicingIn eukaryotes, the poly-A tail is added (100 or more adenines) at the end of the mRNA (polyadenylation)



TranscriptionUntranslated regions– sequences on both end of mRNA, which are not translated into protein– involved in many post-transcriptional regulatory pathways that control mRNA localization, stability and translation efficiency



TranscriptionAfter the transcription (in nucleus), the RNA has to pass to cytosol



TranslationSynthesis of protein from the information contained in a molecule of mRNAOccurs on ribosomes:- Composed of several rRNA and

proteins- 2 subunits – small & large

Initiation- the small ribosomal subunit

binds to the capped end of the mRNA

- Start codon is always methionine (AUG)

- tRNA with methionine binds to the mRNA

- the large ribosomal subunit binds to form the complete initiation complex



TranslationElongation-Translation of codons continues- AA are bound with peptide bond

- Continues till the stop codon is found

Termination- Ribosome reaches one of

the three stop codons: UAA, UAG, UGA

- no tRNA molecule recognizes these codons –translation is complete



Amino acids



Amino acids - mutationsWild Type – Normal SequenceMiss-sense – One base changed

– The sequence coding for a different AANon-sense – One or more bases changed

– Termination of chainSilent – One of more base changes but the result is

the same AAFrameshift Base Deletion – One base removed

– The change of most of the following AAs

Available on URL: http://www.youtube.com/watch?v=41_Ne5mS2ls



Central dogma of molecular biology

DNA RNA PROTEIN

Is it true ??



Reverse transcriptionSynthesis of cDNA (ssDNA) from a RNA templateReverse transcriptase- RNA-dependent DNA

polymerase- DNA-dependent DNA

polymerase- RNase H (a ribonuclease that

degrades RNA in RNA-DNA hybrid structure)

- Ability to unwind DNA-DNA and RNA-DNA duplexes

Key characteristic of retroviruses (but in all living organisms)First detected by Howard Temin and David Baltimore in 1970’ (Nobel Prize in 1975)Retroviruses reverse-transcribe their genome into DNA – incorporation into the host’s genome (integrase)

Video available on URL: http://www.youtube.com/watch?v=68zTyjeUsqY



New dogma of molecular biology

DNA RNA PROTEIN



PCR

Polymerase Chain ReactionDiscovered in 1983 by Kary B. MullisNobel Prize in Chemistry in 1993 (together with Michael Smith)



PCR

In vitro amplification of target DNAUse of DNA polymerase and two specific oligonucleotidesequences (primer), which flank the region of interestA cyclic processEach cycle = double the region marked by primers

The result is up to a billion of copies (amplicons)Primer- Between 18 and 25

nucleotides long- Roughly an equal number of

all four nucleotides- C+G ratio should be 50-60%

Specific or arbitrary



PCRThermus aquaticus

Bacterium living in thermal springs (Lower Geyser Basin)Its polymerase was found to withstand the protein denaturing conditions (95°C)Enabled the PCR



PCR - What do I need?Components:

Template DNAdNTPsPrimers

- Forward- same sequence as

the upper strand- lower strand is a

template - Reverse

- sequence complementary to the upper strand, in a reverse orientation

Polymerase

Reaction buffer- pH 8.3- MgCl2- KCl, Tris-HCl- Stabilisators

H2O (sterile)



PCR - phases

Initial den. - [Denaturation-Annealing-Extension]30x - Final ext.

Initial denaturation- Uncoiling of the strands

Denaturation- Usually at 94-98°C for 30-60 seconds

Annealing- About 40-65°C for usually 30 s- The higher the CG content, the higher the temperature- Generally should be 5°C lower than a melting temperature (Tm)

Extension- Usually 72°C, duration depends on the speed of the polymerase

Final extension- 72°C for about 5 minutes- Full synthesis of all products



PCRReaction volume is usually 20 – 50 µl (microtubes) Thermal cycler



PCR electrophoresisSeparation of PCR

products on agarose gelDNA has a negative electric chargePCR product moves from –to +Separation according to the length of the product (number of bp)Gel dye (EtBr, SYBR®Green…)Sample dyeSize marker (ladder)



Real Time PCR- Quantitative PCR- Estimation of a quantity of PCR product in a real time- Absolute or relative quantification- Use of standard dilutions- Measuring of fluorescence of SYBR®Green (intercalary dye)

Video available on URL: http://www.youtube.com/watch?v=2KoLnIwoZKU



References

This presentation is available on:https://netstorage.czu.cz/NetStorage/http://www.its.czu.cz/cs/?r=841

Alberts B. a kol.: Základy buněčné biologie. Úvod do molekulárníbiologie buňky. 2. vyd. Espero Publishing. Ústí nad Labem 2005.

Rosypal S.: Úvod do molekulární biologie. Čtvrté inovované vydání. Brno: Prof. RNDr. Stanislav Rosypal, DrSc., Brno, 2006.

URL: biology.about.com

URL: http://www.nature.com/scitable



Elaboration of this presentation was financially supported by thegrant of Fund of development of universities FRVŠ 1940/2012 ´Vytvoření laboratorních úloh z molekulární biologie pro praktickou výuku v předmětu „Seed productionand plant breeding’’

Tato prezentace byla vytvořena za finanční podpory grantu FRVŠč.1940/2012 ´Vytvoření laboratorních úloh z molekulární biologie pro praktickou výuku v předmětu „Seed productionand plantbreeding’’



Piece of all of us – one part of human genome



Thank you for your attention!

introduction to molecular biology and basic terminology

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