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Home , Molecular biology, Polyadenylation

FACULTY OF TROPICAL CZECH UNIVERSITY OF SCIENCES breeding and genetic resources conservation AGRISCIENCES PRAGUE

Basic terminology & Introduction to molecular FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES and genetic resources conservation AGRISCIENCES PRAGUE

Basic terminology ¾ – complete set of in an ¾ Chromosomes – the nucleoprotein that carry the genetic information ¾ – the determinant of an observable trait or characteristic of an organism (pigmentation) – the DNA sequence that determines the chemical of a specific polypeptide or RNA molecule ¾ – a viable DNA coding that occupies a given locus (position) on a chromosome – specific form of a gene (blue flower…) FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Basic terminology ¾ – highly alkaline found in eukaryotic nuclei that order the DNA into structural units – nucleosome – condensation of DNA ¾ Nucleosome – basic repeating structural and functional unit of – contains nine histone proteins and about 166 base pairs (bp) of DNA ¾ Chromatine – resulting DNA- complex FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Gene structure ¾ – ¾ – non-coding region ¾ Both are transcribed into pre-mRNA, but only are translated into proteins FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

DNA RNA ¾ Deoxyribonucleic acid ¾ Ribonucleic acid ¾ 2’-deoxy-D-ribose ¾ D-ribose ¾ ¾ Adenine ¾ ¾ Guanine ¾ ¾ Cytosine ¾ Thymine ¾

-Monomer units of nucleic acids are (sugar + base + phosphate) -Each is a phosphate ester of a corresponding nucleoside (sugar and base) -Base order is called sequence FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

DNA Discovered in 1953 by and (Nobel prize in or 1962 together with ) FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

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: ‰ and hydratation ‰ DNA sequence ‰ Presence of special proteins - DNA is compacted more than a thousandfold - Antiparallel strands FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

DNA structure FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

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 FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Types of DNA

¾ According to the number of the ¾ According to the shape: strands: Circular - dsDNA - Covalently Closed Circle -ssDNA (CCC Dna) - Open circle (OC Dna) ¾ According to the location in the cell: Linear Nuclear (nDNA) Non-nuclear - Mitochondrial (mtDNA) - Chloroplast (cpDNA) -Plasmide FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Enzymes influencing DNA

Deoxyribonuclease I (Dnase I) DNA ligase ¾ Binds to the minor groove ¾ Joins Okazaki fragments ¾ Nonspecifically cleaves DNA during replication molecule to small pieces ¾ Completes short-patch DNA ¾ Releases di-, tri- and synthesis occurring in DNA products repair process FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Enzymes influencing DNA Restriction endonucleases

¾ Obtained from (protection against viral infections – non-methylated sequences) ¾ Cleaves both DNA strands in highly specific points ¾ Recognize palindrome (base sequence that reads the same in one direction on one strand as it does the other direction on the other strand) ¾ Create blunt or sticky ends FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Topoisomerases ¾ One of the components of chromatin ¾ Interconvert different topological states of DNA ¾ Change the level of supercoiling ¾ Topoisomerase I cleaves one strand ¾ Topoisomerase II cleaves both strands FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

RNA ¾ Multiple roles in cells ¾ Catalyzators (protein synthesis, splicing…) ¾ Scaffold subcellular structures ¾ Regulation of ¾ Key component for the protein systhesis FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE Types of RNA Messenger RNA (mRNA) Transfer RNA (tRNA) ¾ Conveys sequence information ¾ Brings activated molecules of from the genome to the protein amino acids to synthetic apparatus ¾ Contains anticodon ¾ Consists of codons ¾ 2 functions: (Sequence of three adjacent ¾ Activation (by esterification) of nucleotides constituting the genetic amino acids code that specifies the insertion of an amino acid in a specific structural ¾ Adaptor molecules (for every position in a polypeptide chain during AA there is at least one tRNA) the synthesis of proteins) ¾ Synthesized complementary and antiparallel to the template strand FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE Types of RNA

Ribosomal RNA (rRNA) ¾ Decodes mRNAs into amino acids (fabricates polypeptides) ¾ 2 subunits: the large (LSU, 60S) and the small one (SSU, 40S) ¾ SSU protein, initiation factors and the initiator tRNA, binds to AUG in the 5' region of the mRNA FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

DNA replication ¾ Ensures the transmission of genetic information to daughter cells ¾ Every new cell contains one strand from the mother cell and one new-synthesized strand ¾ Necessity of a primer - a short sequence of RNA from which DNA replication can initiate ¾ OriC – initiation of the replication ¾ Carries out in 5’-3 direction, but on both strands ¾ of many enzymes ¾ Okazaki fragment – short fragment of DNA synthesized on lagging strand FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Replication – enzymes

¾ DNA – separation of ¾ DNA α – two strands ¾ 5' to 3' DNA-dependent DNA ¾ DNA single-stranded binding polymerase proteins – stabilisation of the ¾ 5' to 3' DNA-dependent RNA single strand created by polymerase () helicase ¾ DNA Pol. δ – ¾ DNA gyrase – catalyses the ¾ 3' to 5' exonuclease (proof- formation of negative supercoil reading activity) ¾ Primase – synthetises RNA ¾ 5' to 3' DNA polymerase primer ¾ DNA Pol. ε – ¾ 5' to 3' DNA-dependent DNA polymerase ¾ 3‘ to 5' exonuclease activity ¾ Ligase – binds the lagging strand FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE Replication

Video available on URL: http://www.youtube.com/watch?v=teV62zrm2P0 FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Transcription ¾ mRNA contains the message for ¾ Transcribing of genetic one gene (), or for information from DNA to RNA several adjacent genes ¾ Catalyzed by RNA polymerase II (prokaryotes) (RNAP II) ¾ Starts at (TATA box) – ¾ DNA contains many genes lined binding site for RNAP and up one after another factors FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Transcription

¾ DNA is unwound: ¾ Carries on in 5’-3’ ¾ (untranscribed) direction ¾ Antisense strand (3’-5’; transcribed) ¾ Ends at FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Transcription ¾ Whole gene ranscribed to so- ¾ Activity of small nuclear called pre-mRNA (precursor ribonucleoproteins () – mRNA) intron forms a loop and is cut off ¾ Methyl guanine (“cap”) added at ¾ Remaining exons joined together the beginning of the pre-mRNA – RNA splicing ¾ Non-coding sequences () ¾ In eukaryotes, the poly-A tail is have to be cut off added (100 or more ) at the end of the mRNA () FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Transcription ¾ Untranslated 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 efficiency FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Transcription ¾ After the transcription (in nucleus), the RNA has to pass to FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Translation ¾ Synthesis of protein from the ¾ Initiation information contained in a - the small ribosomal subunit molecule of mRNA binds to the capped end of the ¾ Occurs on ribosomes: mRNA - Composed of several rRNA and - Start codon is always proteins methionine (AUG) - 2 subunits – small & large - tRNA with methionine binds to the mRNA - the large ribosomal subunit binds to form the complete initiation complex FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Translation

¾ Elongation ¾ Termination -Translation of codons continues - reaches one of the three stop codons: - AA are bound with peptide bond UAA, UAG, UGA - no tRNA molecule recognizes these codons – translation is complete

- Continues till the is found FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Amino acids FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Amino acids -

¾ Wild Type – Normal Sequence ¾ Miss-sense – One base changed – The sequence coding for a different AA ¾ Non-sense – One or more bases changed – Termination of chain ¾ Silent – One of more base changes but the result is the same AA ¾ Frameshift Base Deletion – One base removed – The change of most of the following AAs

Available on URL: http://www.youtube.com/watch?v=41_Ne5mS2ls FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Central dogma of DNA RNA PROTEIN

Is it true ?? FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Reverse transcription

¾ Synthesis of cDNA (ssDNA) from ¾ Key characteristic of a RNA template retroviruses (but in all living ) ¾ ¾ First detected by Howard - RNA-dependent DNA Temin and in polymerase 1970’ (Nobel Prize in 1975) - DNA-dependent DNA ¾ Retroviruses reverse- polymerase transcribe their genome into - RNase H (a that DNA – incorporation into the degrades RNA in RNA-DNA hybrid structure) host’s genome () - Ability to unwind DNA-DNA and RNA-DNA duplexes

Video available on URL: http://www.youtube.com/watch?v=68zTyjeUsqY FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

New dogma of molecular biology

DNA RNA PROTEIN FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

PCR

¾ Polymerase ¾ Discovered in 1983 by Kary B. Mullis ¾ Nobel Prize in in 1993 (together with Michael Smith) FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

PCR

¾ In vitro amplification of target DNA ¾ The result is up to a billion of ¾ Use of DNA polymerase and two copies () specific oligonucleotide ¾ Primer sequences (primer), which flank - Between 18 and 25 the region of interest nucleotides long ¾ A cyclic process - Roughly an equal number of ¾ Each cycle = double the region all four nucleotides marked by primers - C+G ratio should be 50-60% ¾ Specific or arbitrary FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

PCR ¾ Bacterium living in thermal springs (Lower Geyser Basin) ¾ Its polymerase was found to withstand the protein denaturing conditions (95°C) ¾ Enabled the PCR FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

PCR - What do I need? ¾ Components: ¾ Reaction buffer ¾ Template DNA -pH 8.3 ¾ dNTPs -MgCl2 ¾ Primers - KCl, -HCl - Forward - Stabilisators - same sequence as ¾ H O (sterile) the upper strand 2 - lower strand is a template - Reverse - sequence complementary to the upper strand, in a reverse orientation ¾ Polymerase FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

PCR - phases

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

¾ Initial denaturation - Uncoiling of the strands ¾ Denaturation - Usually at 94-98°C for 30-60 seconds ¾ 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) ¾ Extension - Usually 72°C, duration depends on the speed of the polymerase ¾ Final extension - 72°C for about 5 minutes - Full synthesis of all products FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

PCR Reaction volume is usually 20 – 50 µl (microtubes) Thermal cycler FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

PCR

¾ Separation of PCR products on gel ¾ DNA has a negative ¾ PCR product moves from – to + ¾ Separation according to the length of the product (number of bp) ¾ Gel dye (EtBr, SYBR®Green…) ¾ Sample dye ¾ Size marker (ladder) FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

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 FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

References

Alberts B. a kol.: Základy buněčné biologie. Úvod do molekulární biologie 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

This presentation is available on: https://netstorage.czu.cz/NetStorage/ http://www.its.czu.cz/cs/?r=841 FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Elaboration of this presentation was financially supported by the grant 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 FRVŠ č.1940/2012 ´Vytvoření laboratorních úloh z molekulární biologie pro praktickou výuku v předmětu „Seed productionand plant breeding’’ FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Piece of all of us –one part of human genome FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE

Thank you for your attention!