Seminarski rad 1: Opisati postupak kloniranja gena organizma
iji genom jo uvek nije sekvenciran
GRUPA 1: studenti sa parnim rednim brojevima na listi Veliina nepoznatog gena oko 40 Kb Pojedini delovi gena koji kodiraju odreene proteinske domene pokazuju visok stepen homologije na nukleotidnom nivou sa odgovarajuim regionima ortolognih gena ije su sekvence poznate i deponovane Navesti ta je potrebno za postupak kloniranja Dati detaljan prikaz eksperimenta kloniranja
GRUPA 2 : studenti sa neparnim rednim brojevimana listi Veliina nepoznatog gena oko 5 kb Gen pokazuje izuzetno visok stepen homologije na nukleotidnom nivou sa ortolognim genima ije su sekvence poznate i deponovane Navesti ta je potrebno za postupak kloniranja Dati detaljan prikaz eksperimenta kloniranja
Seminarski rad otprintovan na najvie 2 strane formata A4 sa imenom studenta, brojem indeksa i potpisom dostaviti na poetku predavanja koje e biti odrano 11.04.2013.g. Organism Genome Size (Mb) Gene Number Hepatitis D virus 0.0017 1 Hepatitis B virus 0.0032 4 HIV-1 0.0092 9 Bacteriophage l 0.0485 80 Escherichia coli 4.6392 4400 S. cerevisiae (yeast) 12.155 6300 C. elegans (nematode) 97 19000 D. melanogaster (fruit fly) 137 13600 Mus musculus (mouse) 3000 20000-30000 Homo sapiens (human) 3000 20000-30000 1 Mb = 1 million base pairs (for double-stranded DNA or RNA) The genomes of prominent organisms E.coli genome CIRCULAR MAP Genetic map: linkage map of known genes or functional sites Often given in minutes (the time which it takes to each male chromosome to move into female cell) Physical: restriction sites of mapped genes
Organisation of E.coli genome A prokaryotic genome has to squise into relatively tiny space with the help of DNA binding proteins that package the genome in an organised fashion
The circular genome is supercoiled: in a typical prokaryote the genome is a single circular DNA molecule is organized in NUCLEOID
The protein component of nucleoid include DNA gyrase and DNA topoisomerase I- the two enzymes that are primarly responsible for maintaining the supercoiled state
A set of at least four additional proteins are beleived to have a more specific roles in packaging of bacterial DNA
The circular E. coli chromosome has a circumference of 1.6 mm (E.coli cell has dimension 1.0 x 2.0 mm) Remove of a few turns of the double helix (underwinding) results in negative supercoiling NUCLEOID Bacteria genome is a nucleoid Although bacteria do not display structure with the distinct morphological features of the eucaryotic chromosomes, bacteria genomes are organized into definite bodies-NUCLEOID. Protein core Between 50 and 100 supercoiled loops of DNA radiate from the central protein core Structures of new Rhs elements Unique DNA sequences from the E. coli K-12 chromosomal framework are designated ORFs f256, o205, and o77 (2). The Rhs elements are aligned so that the start codon of the core ORF is base 1. RhsE and RhsH of ECOR-45 are linked in tandem (indicated by the diagonal dashed line). The core of ECOR-45 RhsG contains a 587-bp deletion, indicated by . Wang etal., JBC Journal of Bacteriology, 1998, 180, 4102-4110. Transposable Elements in Prokaryotes Insertion Sequences (IS)
Noncomposite transposons
Composite Transposons Insertion Sequences (IS) Simplest transposable elements: Segments of DNA in bacteria that can move from one position to another.
Normal constituents of bacterial chromosome and plasmids.
Cause increase in the size of the genome
IS elements only contain genes required to mobilize the element and insert the element at a new location.
When IS elements transpose, promoters within IS elements themselves may alter expression of nearby genes. Insertion Sequences: IS Insertion elements are mobile genetic elements that occasionally insert into chromosomal sequences, often disrupting gene.
IS are a special class of transposable elements found in prokaryotes (studied extensively in Escherichia coli K12)
They usually range in size from 1 to 2 kilobasepairs (kb) and contain perfect or nearly perfect inverted terminal repeat sequences. These terminal repeats likely are recognition sites for an enzyme responsible for the insertion.
Mobility of the element depends only on the element itself; it is an autonomous element. Thus, it must carry the coding ability for the transposase recognizing the inverted terminal repeats.
Insertion Sequences (IS)
The terminal sequences flank a unique central sequence with at least one long open reading frame coding, presumably, for the transposase protein: All IS elements are made up of transposae gene flanked by inverted repeats (IRs).
IS1 first identified in E. colis glactose operon is 768 bp long and is present with 4- 19 copies in the E. coli chromosome
Ends of all known IS elements show inverted terminal repeats (ITRs)
Mechanizm of IS insertion Staggered cut made at target site IS element inserted, joined to single stranded ends: DNA Pol and DNA ligase fills in gaps. Produced target site duplication flanking the IS element. The direct repeats externally flanking the inverted repeats are not part of the insertion sequence. Instead, they are chromosomal sequences that become duplicated upon insertion, with one copy at each end; this is called target-site duplication. Orientation of IS Elements: IS elements can insert in any orientation.
Composite Transposons Created when two IS elements insert near each other. The region between them can then be transposed by joint action of flanking IS elements (i.e. two IS elements "capture" a DNA sequence and endow it with the ability to transpose). In many cases, these "captured" sequences are antibiotic resistance genes. These transposons can jump onto conjugation plasmids and be transferred from one strain or even one species to another. This is of great medical significance. R-factors are multidrug-resistance plasmids containing multiple transposons bearing different antibiotic resistance genes. Composite transposons: Have two ISs at their ends, the DNA between the two ISs can encode resistance genes or virulence factors. In many cases only one of the two transposases is functional.
Noncomposite transposons Noncomposite transposons combine the qualities of both IS elements and composite transposons. Like IS elements, they have single IR sequences at each end. Like composite transposons, they carry selectable marker genes.
Consists of a transposase gene, plus other genes, flanked by inverted repeat (IR) sequences
IR sequences can form "stem-loop" structures when DNA containing transposon is denatured and ssDNA allowed to reanneal. Evolutionary role of insertion sequences
Selfish or parasitic DNA sequences which are maintained in the population, even in the face of adverse natural selection, as a result of their ability to transpose and become horizontally transmitted among strains by means of hitchhiking in plasmids.
Insertion sequences also play an important role in the evolution of transposons and plasmids: One evolutionary implication of insertion sequences derives from their mutagenic activity in causing insertion mutations.
A pair of insertion sequences flanking a central sequence can transpose as a unit, and such composite transposons containing antibiotic-resistance genes, for example,Tn5, Tn9 and TnlO, are well documented
Insertion sequences and transposons can also transpose into plasmids and remold their structure, and in general change the genetic capabilities of plasmids
The distribution of numbers of insertion sequences in the genome is also of some interest in understanding the population dynamics of the elements.
Streptomycetes Comprises a large group of filamentous soil gram- positive bacteria The most important group of industrial microorganisms: producing approximately two-thirds of all known varieties of antibiotics Produces many other useful secondary metabolites and extra cellular enzymes Phenomenon of genetic instability Instability of certain genetic traits: mutations frequently exhibit pleiotropy- several traits being altered at the same time Molecular studies have shown that trait losses are due to large deletions (ten to thousands of KB long) in the chromosomal DNA These large chromosomal DNA (in average 8 MB in size) have terminal inverted repeats Deletions are frequently accompanied by tandem amplifications of up to several hundred copies of specific DNA sequences termed Amplifiable Units of DNA (AUD) This phenomenon is explained by fact that chromosomes of many Streptomycetes species are linear DNA molecules Proteins are covalently attached to the 5-terminal ends of chromosomal DNAs which presumably act as the primers of replication Unstable regions are at the both termini of the chromosome Essentially all deletions involve one, or both, telomeres Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2), 2002, Nature 417, 141 -147 S coelicolor is a soil bacterium that has many different metabolic processes and biotransformations that allow it to live under a wide range of conditions in the soil and use a wide range of metabolic pathways to, for example, degrade insoluble remains of other organisms. S coelicolor is unusual, in that it is a multicellular bacterium which forms into different sorts of 'tissues'. The complexity of its metabolic pathways give rise to two thirds of modern antibiotics, as well as anti-tumour and immuno-suppressant agents. All this complexity arises from the longest known eubacterial genome (8.7 million base pairs and 7,825 genes; about one quarter the number of human genes).
Yeast Genome Yeast Genome: Saccharomyces cerevisiae The entire genome sequence was released in April, 1996 and was the first eukaryotic genome to be sequenced. The haploid genome contains 16 chromosomes ranging in size from 220 to 1500 kb Analysis of the genome reveals 6,183 potential ORFs Compared to the genomes of other organisms, the yeast genes are very efficiently spaced on the 16 chromosomes with a density of 1 gene/2 kb of DNA. Most yeast genes do not contain introns There is little 'junk' DNA in the intergenic regions commonly seen in other eukaryotes. A total genome size of approximately 13,000kb: Chromosomes 85% of the total yeast DNA The 2mm-plasmid accounts for about 5%, Mitochondrial DNA 10% The 6.3 kb 2mm-plasmid plasmid has no known function in yeast other than to replicate, but it is not deleterious to the cell. Each cell contains 60-100 copies of this plasmid. Researchers have taken advantage of this naturally occurring high copy number plasmid to create yeast plasmid vectors containing the 2mm-plasmid origin of replication. Life with 6000 genes Science,1996, Vol. 274, p546 Reports on the complete sequencing of the genome of the yeast Saccharomyces cerevisiae. The sequence of 12,068 kilobases defining: 6183 potential ORFs of at least 100 amino acids in length. However, there are likely additional ORFs smaller than 100 amino acids and not all of the 6,183 potential ORFs are protein encoding genes. 5885 potential protein-encoding genes 140 genes specifying ribosomal RNA; 40 genes for small nuclear RNA molecules; 275 transfer RNA genes Providing of information about the higher order organization of yeast's 16 chromosomes and allowance of some insight into their evolutionary history The total number of real protein-coding genes It has been proposed a revised yeast gene catalog consisting of 5538 ORFs 100 amino acids. This reflects the proposed elimination of 503 ORFs.
Only two-thirds of these have been experimentally validated (known), and the remaining ~2000 ORFs are currently annotated as hypothetical. The total number of real protein-coding genes has been a subject of considerable debate, with estimates ranging from 4,800 to 6,400 genes. Saccharomyces cerevisiae complete genome The size of DNA in each chromosome of the yeast S. cerevisiae. The elements are some 6 kb flanked by long terminal repeats (LTRs; some 300 bp in length), which are named delta (for Ty1/2), sigma (for Ty3) and tau (for Ty4). Ty elements resemble retroviral genomes in yet other aspects: there are two overlapping (in a +1 mode) open reading frames. ORF TyA encodes the gag (envelope) protein of the virus-like particles; TyB provides a polycistronic message the product of which is processed by the endogenous Asp-type protease to yield a Ty integrase and a retro transcriptase. Though the elements share a relatively high degree of homology, Ty1/2/4 belong to the 'copia' class of retroelements, while Ty3 is a member of the 'gipsy' family. The majority of the Ty elements were found to transpose upstream of tRNA genes, in a sequence non-specific manner. These sites appear to represent less harmful targets than genes or other intergenic regions. Retrotransposons in yeast
-Ty1- Ty5. -Ty1 through Ty4 have been identified to retrotranspose via an RNA intermediate -Ty5 does appear to be no longer capable of transposition
The genome sequence of Schizosaccharomyces pombe Nature. 2002 Feb 21;415(6874):871-80. The genome of fission yeast (Schizosaccharomyces pombe) contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824 The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns. 50 genes have significant similarity with human disease genes; half of these are cancer related. Highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing are identified. These genes may have originated with the appearance of eukaryotic life. Few conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.
Genetic mapping A pair of homologous chromosomes can exchange parts by crossing-over
Recombination produces genotypes with new combinations of parental alleles
Two genes close together on the same chromosome pair do not assort independently at meiosis
Gene loci on chromosome can be mapped by measuring the frequencies of recombinants produced by crossing-over Meiotic recombination Process that generates a haploid product with genotype that differs from both haploid genotypes that constituted the meiotic diploid cell
The product of meiosis generated by recombination is called RECOMBINANT Haplotype Recombination will rarely separate loci which lie close together on a chromosome
A set of alleles on the small chromosomal segment tend to be transmitted as a block through a pedigree
Such block of alleles is known as HAPLOTYPE
Haplotypes mark recognizable chromosomal segments which can be traced through pedigrees and through population Genetic distance The recombination fraction is a measure of the distance between two loci
The recombination fraction define genetic distance Two loci which show 1% recombination are defined as being 1 CENTIMORGAN apart on a genetic map a) b) a b A B A B a b a B A b a b A B a b A B a b a B A b Heterozigotni roditelj Produkti mejotike rekombinacije Dobijena frekvenca 45% 45% 5% 5% 90% roditeljski 10% rekombinant Heterozigotni roditelj Produkti mejotike rekombinacije Oekivana frekvenca 25% 25% 25% 25% 50% roditeljski 50% ne-roditeljski Nezavisna segregacija Genetic markers Mendelian characters which are sufficiently polymorphic to give a reasonable chance that randomly selected person will be heterozygous Highly polymorphic and informative Randomly distributed Easy to score High PIC values PIC (polymorphism information content) a measure of the informativness of genetic marker STS (sequence tagged site): any piece of DNA whose sequence is known and for which a specific PCR assay has been designed
EST (expressed tagged site): a short sequence of cDNA for which a PCR assay is available RFLP
Restriction Fragment Length Polymorphism A polymorphism due to difference in size of allelic restriction fragments as a result of restriction site polymorphism Effects at the protein level Effects at the DNA level Normal red blood cells (top) and sickle cells (bottom) There are effects at the cellular level. When red blood cells carrying mutant hemoglobin are deprived of oxygen, they become sickle-shaped instead of the usual round shape (see picture). This shape can sometimes interrupt blood flow. There are negative effects at the whole organism level. Under conditions such as high elevation and intense exercise, a carrier of the sickle cell allele may occasionally show symptoms such as pain and fatigue. There are positive effects at the whole organism level. Carriers of the sickle cell allele are resistant to malaria, because the parasites that cause this disease are killed inside sickle-shaped blood cells. Sickle Cell Anemia RFLP and sickle cell mutation CCTTAGG GGAATCC CCTGAGG GGACTCC CCTTAGG GGAATCC CCTTAGG GGAATCC CCTGTGG GGAAACC CCTTAGG GGAATCC 0.2 KB 1.1 KB 1.3 KB Mst I I Mst I I Mst I I Mst I I Mst I I b a b s RFLP and sickle cell mutation Vezanost markera Kada se ispituje odreeno svojstvo (osobina, oboljenje) prvi korak je odreivanje na kom hromozomu se nalazi traeni genski lokus
To se radi indirektno traenjem neke druge genetike karakteristike (odnosno genetikog markera) koji se nasleuje u korelaciji sa bolesnim alelom
Vezanost je korelacija u nasleivanju nekog RFLP alela (ili nekog drugog genetikog markera) i bolesnog alela Linkage Analyis: analiza vezanosti markera
Tendencija da se markeri na specifinom lokusu nasleuju zajedno kao posledica njihove fizike vezanosti na pojedinanim hromozomima A probe P detects two DNA morphs when DNA is cut by a certain restriction enzyme (RE).
The pedigree of analysis of dominant disease phenotype D shows: -Linkage of the D locus to the RFLP locus in childs 2, 3, 6 and 7 -Absence of linkage in child 8 ????? The detection and inheritance of RFLP The detection and inheritance of RFLP
The pedigree of dominant disease phenotype D shows linkage of the D locus to the RFLP locus.
Only child 8 is recombinant Mapping using microsatellite repeats as molecular markers Disease allele P is probably linked to M Mapping using DNA Fingerprint bands as molecular marker DNA fingerprint The specific pattern of DNA fragments formed when DNA is cut with restriction enzymes, separate and hybridize
Alele P possible linked to C or I A B C D E F G I H F and H: Always inherited together- linked? A and B: in progeny always either A or B- allelic? A and D: Four combinations; A and D, A, D or neither- unlinked? F, H and E: Always either F or H or E- closely linked in trans? DNA profiling In violent crimes such a murder or rape and in paternity (or grand-paternity) cases. Fortunately DNA is quite stable and resistant to degradation. With the latest procedures available sufficient DNA may be obtained from just a single hair follicle.
Autoradiograph from an actual rape case showing the DNA profiles for one VNTR locus. The lanes marked "M" show a "ladder" of DNA fragments of known sizes. These are loaded onto the gel to provide an internal ruler--allowing the sizes of the VNTR alleles to be estimated more accurately. As can be seen there is a match of the DNA profile of defendant 1 and the forensic sample.
LOD SCORE Mera verovatnoe o genetikoj vezanosti izmeu lokusa