FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE Basic terminology & Introduction to molecular biology FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE Basic terminology ¾ Genome – complete set of genes in an organism ¾ Chromosomes – the nucleoprotein structures that carry the genetic information ¾ Gene – 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 molecule ¾ Allele – 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 Histone – highly alkaline proteins found in eukaryotic cell nuclei that order the DNA molecules into structural units – nucleosome – condensation of DNA ¾ Nucleosome – basic repeating structural and functional unit of chromatin – contains nine histone proteins and about 166 base pairs (bp) of DNA ¾ Chromatine – resulting DNA-protein complex FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE Gene structure ¾ Exon – coding region ¾ Intron – non-coding region ¾ Both are transcribed into pre-mRNA, but only exons 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 Adenine ¾ Adenine ¾ Guanine ¾ Guanine ¾ Cytosine ¾ Cytosine ¾ Thymine ¾ Uracil -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 FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation AGRISCIENCES PRAGUE DNA Discovered in 1953 by James Watson and Francis Crick (Nobel prize in Physiology or Medicine 1962 together with Maurice Wilkins) 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: Ions 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 oligonucleotide 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 bacteria (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 gene expression 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 ribosomes 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 Interaction 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 helicase – separation of ¾ DNA Polymerase 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 (primase) 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 (eukaryotes), or for information from DNA to RNA several adjacent genes ¾ Catalyzed by RNA polymerase II (prokaryotes) (RNAP II) ¾ Starts at promoter (TATA box) – ¾ DNA contains many genes lined binding site for RNAP and up one after another transcription 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 Sense strand (untranscribed) direction ¾ Antisense strand (3’-5’; transcribed) ¾ Ends at terminator 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 (snRNPs) – 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 (introns) ¾ In eukaryotes, the poly-A tail is have to be cut off added (100 or more adenines) at the end of the mRNA (polyadenylation) 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 translation efficiency FACULTY OF TROPICAL CZECH UNIVERSITY OF LIFE SCIENCES Plant breeding and genetic resources conservation