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Lecture 4. Genome Genome - the complete set of chromosomes inherited from a single parent; the complete DNA component of an individual. A chromosome The DNA molecule may be circular or linear, and can be composed of 10,000 to 1,000,000,000 base pairs. Typically eukaryotic cells have large linear chromosomes and prokaryotic cells have smaller circular chromosomes. In eukaryotes, nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. Chromosomes may exist as either duplicated or unduplicated—unduplicated chromosome is linear DNA molecule, whereas duplicated chromosome contains two copies of DNA joined by a centromere. In prokaryotes DNA is usually arranged as a circle, which is tightly coiled in, sometimes accompanied by smaller, circular DNA molecules called plasmids. The small circular DNA molecules are also found in mitochondria and chloroplasts, reflecting their bacterial origins. The simplest genomes are found in viruses: these DNA or RNA molecules are short linear or circular that often lack structural proteins. Eukaryotic Chromosome Structure Eukaryotic chromosomes consist of condensed DNA and associated proteins called histones and non-histons. DNA-protein complex is named chromatin. The fundamental unit of chromatin is the nucleosome Histones are basic proteins that form the nucleosomes. The nucleosomes are then arranged in a coiled pattern to produce a chromatin fiber. This fiber is further compacted with non-histones by looping to produce looped domains. The looped domains are coiled and compacted to produce chromosomes. Nucleosome Structure of Chromosomes Nucleosome - simplest packaging structure of eukaryotic chromosomes; the nucleosome consists of about 200 bp of DNA wrapped around a histones. 146 bp DNA is wrapped around an octamer that contains two copies of histone proteins H2A, H2B, H3 and H4. The remaining bases link to the next nucleosome; this structure causes negative super coiling. The 30 nm fiber is the next level of organization of the chromatin. This appears to be a solenoid structure with about six nucleosomes per turn. The stability of this structure requires the presence of histone H1. Fig 6. The levels of organization of the chromatin: the nucleosomes’ chain. Chromatin structure. The final level of packaging is characterized by the 700 nm structure seen in the metaphase chromosome. This appears to be the result of extensive looping of the DNA in the chromosome. Chromatin - the unit of analysis of the chromosome. Heterochromatin is DNA that is coiled and condensed. In this state, it is not transcribed. Euchromatin is less condensed chromatin, it is transcribed. During the process of cell division, the DNA becomes tightly coiled, forming structures called chromosomes. They are doubled after replication in S phase and consist of two chromatids joining together be the centromere. Centromeres and Telomeres Centromeres and telomeres are two essential features of all eukaryotic chromosomes. Centromeres are required for the segregation of the chromosomes during cell division. Telomeres - the region of DNA at the end of linear eukaryotic chromosome; required for the replication and stability of the chromosome. The telomeric DNA has heterochromatin and contains direct tandemly repeated sequences (e.g., TTAGGG). Classes of Eukaryotic DNA Sequences Coding and Non Coding DNA Sequences Less than 5% of eukaryotic DNA codes for proteins. Approximately 1.5% of human DNA codes for protein. The function of the remaining DNA is not known but perhaps much of it has no function. Essential Conserved Non Coding DNA Sequences These DNA sequences do not code for proteins and include: 1) promoters (sites that bind RNA polymerases), 2) regulatory elements (enhancers, silencers, and locus control regions LCRs) that bind regulatory proteins, 3) the origin of replication (sites that bind the DNA replication complex), 4) the centromeric and the telomeric DNA. Analysis of DNA Sequences in Eukaryotic Genomes The technique that is used to determine the sequence complexity of any genome involves the denaturation and renaturation of DNA. DNA is denatured by heating which melts the H-bonds and renders the DNA single-stranded. If the DNA is allowed to cool slowly, sequences that are complementary will find each other and eventually base pair again (DNA renaturates or reanneals). Genomes that contain different classes of sequences reanneal in a different manner. The first sequences to reanneal are the highly repetitive sequences because so many copies of them exist in the genome. The second portion of the genome to reanneal is the middle repetitive DNA, and the final portion to reanneal is the single copy DNA. Classes of Eukaryotic DNA Sequences Single copy sequences Found once or a few times in the genome Reanneal very slowly The sequences which encode functional genes fall into this class The genes have exons and introns Repeated DNA sequences are found more than once in the genome of the species. Middle repetitive Sequences (DNA) Found from 10 to 1000 times in the genome Examples include rRNA, tRNA genes and histones genes Middle repetitive DNA can vary from 100-300 bp to 5000 bp and can be dispersed throughout the genome (they are named SINES and LINES). Highly Repetitive Sequences 10-25% of eukaryotic DNA consists of sequences of 5 to 10 nucleotides repeated 100,000 to 1,000,000 times. They are minisatellites and microsatellites DNA. Reanneal very rapidly They do not transcribe These sequences are found from 100,000 to 1 million times in the genome These sequences are found in heterochromatin, centromeric and telomeric DNA Tend to be arranged as a tandem repeats; o ATTATA ATTATA ATTATA // ATTATA Extra chromosomal DNA Mitochondrial DNA In animals the mitochondrial genome is typically a single circular chromosome and mitochondrial DNA lacks introns; however, introns have been observed in mitochondrial DNA of yeast and protists. There is a very high proportion of coding DNA and an absence of repeats in mitochondrial genome. Not all proteins necessary for mitochondrial function are encoded by the mitochondrial genome; most proteins are coded by genes in the cell nucleus and are imported into the mitochondrion. The human mitochondrial genome is a circular DNA molecule of about 16 000 bp. It encodes 37 genes: 13 for subunits of respiratory complexes I, III, IV and V; 22 genes for mitochondrial tRNA (for the 20 standard amino acids, plus an extra gene for leucine and serine), and 2 for rRNA. One mitochondrion can contain two to ten copies of its DNA. Genetic code in mitochondria Mitochondrial genes use an alternative mitochondrial code. Many slight variants have been discovered. Further, the AUA, AUC, and AUU codons are all start codons. Replication and inheritance Mitochondria divide by binary fission similar to bacterial cell division. In many singlecelled eukaryotes, their growth and division is linked to the cell cycle. In other eukaryotes (in humans for example), mitochondria may replicate their DNA and divide in response to the energy needs of the cell. When the energy needs of a cell are high, mitochondria grow and divide. When the energy use is low, mitochondria become inactive. Mitochondria and the mitochondrial DNA are inherited down the female line, known as maternal inheritance. This mode is seen in most organisms including humans. Mitochondrial diseases Damage and dysfunction in mitochondria is an important factor in a wide range of human diseases. Mitochondrial disorders often present as neurological disorders, but can manifest as myopathy, diabetes and multiple endocrinopathy. Prokaryotes Genome Chromosomes in prokaryotes The chromosome of prokaryotes consists of a single circular double-stranded DNA. It is not condensed into chromosomes as in eukaryotes. Structure in sequences There is a very high proportion of coding DNA and an absence of repeats in bacteria genome. Bacteria typically have a single origin of replication. The genes in prokaryotes are often organized in operons, and do not contain introns. DNA packaging Prokaryotes DNA is organized into a structure called the nucleoid. Histone-like proteins associate with the bacterial chromosome. Prokaryotic chromosomes and plasmids are supercoiled double-stranded DNA. The DNA must first be released into its relaxed state for access for transcription, regulation, and replication. Bacterial chromosomes tend to be tethered to the plasma membrane of the bacteria. Extra chromosomal Prokaryotic DNA Plasmids A plasmid is an extra chromosomal circular and double-stranded DNA molecule which is capable of replicating independently from the chromosomal DNA. Plasmids usually occur in bacteria, but are sometimes found in eukaryotic organisms (e.g., in Saccharomyces cerevisiae). Plasmids are often associated with conjugation, a mechanism of gene transfer. Plasmids may carry genes that provide resistance to naturally occurring antibiotics. Plasmids are now being used to manipulate DNA and may be a tool for genetic engineering techniques. Types of plasmids Fertility-F-plasmid which contain tra-genes (transfer). They are capable of conjugation and help bacteria produce pili. Resistance- R-plasmids, which contain genes that can build a resistance against antibiotics or poisons. Col-plasmids, which contain genes that determine the production of bacteriocins, proteins that can kill other bacteria cells. Degradative plasmids, which enable the digestion of unusual substances (toluene or salicylic acid). Virulence plasmids, which turn the bacterium into a pathogen (one that causes disease). Mobile genetic elements Transposons and retrotransposons Transposons are discrete segments of DNA capable of moving through the genome of their host. There are two types of transposons: Class I retrotransposons move via an RNA intermediate by using RNApolymerase and then reverse transcriptase to produce cDNA (copy DNA). Class II DNA transposons move via "cut-and-paste" mechanism by using of their own enzyme transposase. Fig.7. Transposon structure: transposons are discrete segments of DNA capable of moving through the genome. Both sides of transposon flanking by inverted sequences (IS). Gene for transposition codes for enzyme transposase. Structural genes often codes for antibiotic resistance for bacteria cell.