1. Molecular Genetics Introduction to
Structures and Functions of Nucleic Acids
Organization and Content of Genomes
DNA Replication
The Mutability and Repair of DNA
DNA Recombination
Transcription and RNA Processing
Translation
Regulation of Gene Expression
Techniques of Molecular Genetics
Figure 6-2 The pathway from DNA to protein. The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells.
Studiju materiāli / MolekularasBiologijas / Ievads MolGen / EN

2. the molecule that encodes genetic information in all cells
DNA
the molecule that encodes genetic information in all cells
[many viruses store the genetic information in the RNA molecule]
GENOME
the complete haploid set of an organism’s DNA
(or the whole genetic information of an organism)
divided: physically – into CHROMOSOMES
functionally – into GENES and other elements
CHROMOSOME
a discrete unit of the genome carrying single DNA molecule that contains many genes
GENE
a segment of DNA specifying a protein or noncoding (nc) RNA

3. Prokaryotes typically have one chromosome
Eukaryotes always have many chromosomes
Figure 7-1. Comparison of typical prokaryotic and eukaryotic cells. (a) The diameter of a typical eukaryotic cell is ~10 mkm. The typical prokaryotic cell is ~1 mkm long. (b) Prokaryotic chromosomal DNA is located in the nucleoid and occupies a substantial portion of the internal region of the cell. Unlike the eukaryotic nucleus, the nucleoid is not separated from the remainder of the cell by a membrane. Plasmid DNA is shown in red. (c) Eukaryotic chromosomes are located in the membrane-bound nucleus. Haploid (one copy) and diploid (two copies) cells are distinguished by the number of copies of each chromosome present in the nucleus.
Molecular Biology of the Gene, 5th Edition

4. The chromosome content of the HUMAN GENOME
Figure Human chromosomes. These chromosomes, from a male, were isolated from a cell undergoing nuclear division (mitosis) and are therefore highly compacted. Each chromosome has been "painted" a different color to permit its unambiguous identification under the light microscope. Chromosome painting is performed by exposing the chromosomes to a collection of human DNA molecules that have been coupled to a combination of fluorescent dyes. For example, DNA molecules derived from chromosome 1 are labeled with one specific dye combination, those from chromosome 2 with another, and so on. Because the labeled DNA can form base pairs, or hybridize, only to the chromosome from which it was derived (discussed in Chapter 8), each chromosome is differently labeled. For such experiments, the chromosomes are subjected to treatments that separate the double-helical DNA into individual strands, designed to permit base-pairing with the single-stranded labeled DNA while keeping the chromosome structure relatively intact. (A) The chromosomes visualized as they originally spilled from the lysed cell. (B) The same chromosomes artificially lined up in their numerical order. This arrangement of the full chromosome set is called a karyotype. (From E. Schröck et al., Science 273:494497, © AAAS.)

5. Eukaryotic organelles also contain genetic information
Genomes, 2nd Edition
Figure 1.1. The nuclear and mitochondrial components of the human genome.

6. Genome size and gene number roughly correlate with organism’s complexity
(modified)

7. Viral genomes are in many forms
A map of the HIV genome
Figure Schematic drawings of several types of viral genomes. The smallest viruses contain only a few genes and can have an RNA or a DNA genome. The largest viruses contain hundreds of genes and have a double-stranded DNA genome. The peculiar ends (as well as the circular forms) overcome the difficulty of replicating the last few nucleotides at the end of a DNA strand (discussed in Chapter 5).
Figure A map of the HIV genome. This retroviral RNA genome consists of about 9000 nucleotides and contains nine genes, the locations of which are shown in green and red. Three of the genes (green) are common to all retroviruses: Gag encodes capsid proteins, Env encodes envelope proteins, and Pol encodes both the reverse transcriptase (which copies the RNA into DNA) and the integrase (which inserts the DNA copy into the host cell genome) (discussed in Chapter 5). The HIV genome is unusually complex, because in addition to the three large genes (green) normally required for the retrovirus life cycle, it contains six small genes (red). At least some of these small genes encode proteins that regulate viral gene expression (Tat and Rev—see Figure 7–103); others encode proteins that modify host cell processes, including protein trafficking (Vpu and Nef) and progression through the cell cycle (Vpr). As indicated by the red lines, RNA splicing (using host cell spliceosomes) is required to produce the Rev and Tat proteins.

8. Bacterial genomes are almost entirely composed of genes
~ 0.2% (9000 bp) of the E. coli chromosome
Genome of E. coli
Figure The genome of E. coli. (B) A diagram of the E. coli genome of 4,639,221 nucleotide pairs (for E. coli strain K-12). The diagram is circular because the DNA of E. coli, like that of other procaryotes, forms a single, closed loop. Protein-coding genes are shown as yellow or orange bars, depending on the DNA strand from which they are transcribed; genes encoding only RNA molecules are indicated by green arrows. Some genes are transcribed from one strand of the DNA double helix (in a clockwise direction in this diagram), others from the other strand (counterclockwise). (A, courtesy of Tony Brain and the Science Photo Library; B, after F. R. Blattner et al., Science 277: , © AAAS.)
Figure Directions of transcription along a short portion of a bacterial chromosome. Some genes are transcribed using one DNA strand as a template, while others are transcribed using the other DNA strand. The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads). Approximately 0.2% (9000 base pairs) of the E. coli chromosome is depicted here. The genes transcribed from left to right use the bottom DNA strand as the template; those transcribed from right to left use the top strand as the template.

9. Mitochondria have cut-down versions of bacterial genomes
The Human mitochondrial genome:
(i) Respiratory complex genes (ii) Ribosomal RNA genes (iii) Transfer RNA genes
Figure The organization of the human mitochondrial genome. The genome contains 2 rRNA genes, 22 tRNA genes, and 13 protein-coding sequences. The DNAs of many other animal mitochondrial genomes have also been completely sequenced. Most of these animal mitochondrial DNAs encode precisely the same genes as humans, with the gene order being identical for animals that range from mammals to fish.

10. Gene density is decreased in more complex organisms
Molecular Biology of the Gene, 6th Edition
Figure 7-2. Comparison of chromosomal gene density for different organisms. A 65-kb region of DNA including the gene for the largest subunit of RNA polymerase (RNA polymerase II for eukaryotic cells) is illustrated for each organism. In each case, the RNA polymerase encoding DNA is indicated in red. Coding DNA for other genes is indicated in green, intron DNA in purple, repeated DNA in yellow, and unique intergenic DNA in grey. Note how the number of genes in the 65-kb region decreases as organism complexity increases.

11. The organization of genes on a human chromosome
Figure The organization of genes on a human chromosome. (A) Chromosome 22, one of the smallest human chromosomes, contains 48 × 106 nucleotide pairs and makes up approximately 1.5% of the entire human genome. Most of the left arm of chromosome 22 consists of short repeated sequences of DNA that are packaged in a particularly compact form of chromatin (heterochromatin), which is discussed later in this chapter. (B) A tenfold expansion of a portion of chromosome 22, with about 40 genes indicated. Those in dark brown are known genes and those in light brown are predicted genes. (C) An expanded portion of (B) shows the entire length of several genes. (D) The intron-exon arrangement of a typical gene is shown after a further tenfold expansion. Each exon (red) codes for a portion of the protein, while the DNA sequence of the introns (gray) is relatively unimportant. The entire human genome (3.2 × 109 nucleotide pairs) is distributed over 22 autosomes and 2 sex chromosomes (see Figures 4-10 and 4-11). The term human genome sequence refers to the complete nucleotide sequence of DNA in these 24 chromosomes. Being diploid, a human somatic cell therefore contains roughly twice this amount of DNA. Humans differ from one another by an average of one nucleotide in every thousand, and a wide variety of humans contributed DNA for the genome sequencing project. The published human genome sequence is therefore a composite of many individual sequences. (Adapted from International Human Genome Sequencing Consortium, Nature 409:860921, 2001.)

12. The sequence content of the HUMAN GENOME
Figure Representation of the nucleotide sequence content of the human genome. LINES, SINES, retroviral-like elements, and DNA-only transposons are all mobile genetic elements that have multiplied in our genome by replicating themselves and inserting the new copies in different positions. These mobile genetic elements are discussed in Chapter 5. Simple sequence repeats are short nucleotide sequences (less than 14 nucleotide pairs) that are repeated again and again for long stretches. Segmental duplications are large blocks of the genome ( ,000 nucleotide pairs) that are present at two or more locations in the genome. The most highly repeated blocks of DNA in heterochromatin have not yet been completely sequenced; therefore about 10% of human DNA sequences are not represented in this diagram. (Data courtesy of E.Margulies)

13. Prokaryotic DNA is floating in cytoplasm
Prokaryotic DNA is floating in cytoplasm Eukaryotic DNA is located within nucleus
Molecular Cell Biology, 4th Edition

14. In nucleus, DNA is associated with proteins: CHROMATIN
Histones
- H1, H2A, H2B, H3, H4
Nonhistone proteins
Figure 4-9. A cross-sectional view of a typical cell nucleus. The nuclear envelope consists of two membranes, the outer one being continuous with the endoplasmic reticulum membrane (see also Figure 12-9). The space inside the endoplasmic reticulum (the ER lumen) is colored yellow; it is continuous with the space between the two nuclear membranes. The lipid bilayers of the inner and outer nuclear membranes are connected at each nuclear pore. Two networks of intermediate filaments (green) provide mechanical support for the nuclear envelope; the intermediate filaments inside the nucleus form a special supporting structure called the nuclear lamina.

15. Chromosomes exist in distinct states during different phases of the cell cycle
Molecular Biology of the Gene, 5th Edition
Figure Changes in chromatin structure. Chromosomes are maximally condensed in M phase and decondensed throughout the rest of the cell cycle (G1, S, and G2 in mitotic cells). Together these decondensed stages are referred to as interphase.

16. Chromosomes exist in distinct states during different phases of the cell cycle
Figure A comparison of extended interphase chromatin with the chromatin in a mitotic chromosome. (A) An electron micrograph showing an enormous tangle of chromatin spilling out of a lysed interphase nucleus. (B) A scanning electron micrograph of a mitotic chromosome: a condensed duplicated chromosome in which the two new chromosomes are still linked together (see Figure 4-22). The constricted region indicates the position of the centromere. Note the difference in scales. (A, courtesy of Victoria Foe; B, courtesy of Terry D. Allen.)

17. Chromosomes have a hierarchic organization
Life The Science of Biology, 7th Edition
Figure 9.6. DNA packs into mitotic chromosome. The nucleosome, formed by DNA and histones, is the essential building block in this highly packed structure. 17

18. The different forms of interphase chromatin
Molecular Biology of the Gene, 5th Edition
The different forms of interphase chromatin
Figure Forms of chromatin structure seen in the EM. (a) Electron micrographs of M phase and interphase DNA show the changes in the structure of chromatin. (b) Electron micrographs of different forms of chromatin in interphase cells show the 30-nm and the 10-nm chromatin fibers (beads on a string).

19. NUCLEOSOME is the basic unit of eukaryotic chromosome structure
Molecular Biology of the Gene, 5th Edition
Figure DNA packaged into nucleosomes. (a) Schematic of the packaging and organization of nucleosomes.

20. NUCLEOSOME = 2x H2A, H2B, H3, H4 + DNA
Figure Structural organization of the nucleosome. A nucleosome contains a protein core made of eight histone molecules. In biochemical experiments, the nucleosome core particle can be released from isolated chromatin by digestion of the linker DNA with a nuclease, an enzyme that breaks down DNA. (The nuclease can degrade the exposed linker DNA but cannot attack the DNA wound tightly around the nucleosome core.) After dissociation of the isolated nucleosome into its protein core and DNA, the length of the DNA that was wound around the core can be determined. This length of 147 nucleotide pairs is sufficient to wrap 1.7 times around the histone core.

21. Histones are small, basic proteins that share a common structural motif - HISTONE FOLD
Molecular Biology of the Gene, 5th Edition
Figure The core histones share a common structural fold. (a) The four histones are diagrammed as linear molecules. The regions of the histone fold motif that form alfa helices are indicated as cylinders. Note that there are adjacent regions of each histone that are structurally distinct including additional alfa helical regions. (b) The helical regions of two histones (here H2A and H2B) come together to form a dimer. H3 and H4 also use a similar interaction to form H3-H4 tetramers.

22. Histone folds bind to each other mediating THE ASSEMBLY OF NUCLEOSOMES
Molecular Biology of the Gene, 5th Edition
Histone folds bind to each other mediating THE ASSEMBLY OF NUCLEOSOMES
Figure The assembly of a nucleosome. The assembly of a nucleosome is initiated by the formations of a H3-H4 tetramer. The tetramer then binds to dsDNA. The H3-H4 tetramer bound to DNA recruits two copies of the H2A-H2B dimer to complete the assembly of nucleosome.

23. Higher order chromatin structure: 10-nm fiber is packed into 30-nm fiber
6-fold compacting
40-fold compacting
Molecular Biology of the Gene, 5th Edition

24. Linker histone H1 and tails of the core histones act to form the 30-nm fiber
Molecular Biology of the Gene, 6th Edition
Figure Histone H1 induces tighter DNA wrapping around the nucleosome. The two illustrations show a comparison of the wrapping of DNA around the nucleosome in the presence and absence of histone H1. One histone H1 can associate with each nucleosome.
Figure Speculative model for the stabilization of the 30-nm fiber by histone amino-terminal tails. In this model, the 30-nm fiber is illustrated using “zigzag” model. Several different tail-histone core interactions are possible. Here, the interactions are shown as between every alternate histone, but they could also be with adjacent or more distant histones.

25. Further compaction involves large loops of 30-nm fiber
1000-fold compacting
Molecular Biology of the Gene, 5th Edition
Figure Higher-order structure of chromatin. (b) A model for the structure of a eukaryotic chromosome shows that the majority of the DNA is packaged into a large loops of 30-nm fiber that are tethered to the nuclear scaffold at their base. Sites of active DNA manipulation (e.g., sites of transcription or DNA replication are in the form of 10-nm fiber or even naked DNA.

26. Interphase chromosomes are ‘fluid’ - REGULATION of chromatin structure
Figure A model for the structure of an interphase chromosome. A section of an interphase chromosome is shown folded into a series of looped domains, each containing perhaps 50, ,000 nucleotide pairs of double-helical DNA condensed into a 30-nm fiber. The chromatin in each individual loop is further condensed through poorly understood folding processes that are reversed when the cell requires direct access to the DNA packaged in the loop. Neither the composition of the postulated chromosomal axis nor how the folded 30nm-fiber is anchored to its is clear. However, in mitotic chromosomes the bases of the chromosomal loops are enriched both in condensins and in DNA topoisomerase II enzymes, two proteins that may form much of the axis at metaphase (see Figure 4-74).

27. Structure and positioning of nucleosomes is determined by non-histone proteins
Figure Irregularities in the 30-nm fiber. This schematic view of the 30-nm fiber illustrates its interruption by sequence-specific DNA-binding proteins. How these proteins bind tightly to DNA is explained in Chapter 7. The interruptions in the 30-nm fiber may be due to regions of DNA that lack nucleosomes altogether or, more probably, to regions that contain altered or remodeled nucleosomes. Regions of chromatin that are nucleosome free or contain remodeled nucleosome can often be detected experimentally by the unusually high susceptibility of their DNA to digestion by nucleasesas compared with the DNA in nucleosomes.

28. Types of changes in chromatin during transcription initiation
Figure Local alterations in chromatin structure directed by eucaryotic gene activator proteins. Histone acetylation and nucleosome remodeling generally render the DNA packaged in chromatin more accessible to other proteins in the cell, including those required for transcription initiation. In addition, specific patterns of histone modification directly aid in the assembly of the general transcription factors at the promoter (see Figure 7-46). Transcription initiation and the formation of a compact chromatin structure can be regarded as competing biochemical assembly reactions. Enzymes that increase, even transiently, the accessibility of DNA in chromatin will tend to favor transcription initiation (see Figure 4-34). 28

29. Histone modifications alter the ‘meaning’ of chromatin
Recombinant DNA, 3rd Edition
Figure Histone modifications. A diagram of the histone octamer and the multiple sites on the histone tails where different covalent histone modification can occur. 29

30. Specific meanings of the histone modifications: HISTONE CODE
Figure Some specific meanings of the histone code. (A) The modifications on the histone H3 N-terminal tail are shown, repeated from figure (B) The H3 tail can be marked by different combinations of modifications that convey a specific meaning to the stretch of chromatin where this combination occurs. Only a few of the meanings are known, including the four examples shown. To focus on just one example, the trimethylation of lysine 9 attracts the heterochromatin-specific protein HP1, which induces a spreading wave of further lysine 9 trimethylation followed by further HP1 binding, according to the general scheme that will be illustrated shortly (see Figure 4-46). Not shown is the fact, as just implied (see Figure 4-43), reading the histone code generally involves the joint recognition of marks at other sites on the nucleosome along with the indicated H3 tail recognition. In addition, specific levels of methylation (mono-, di-, or tri-methyl groups) are required, as in Figure 4-42.

31. There are two types of interphase chromatin
Molecular Biology of the Cell, 5th Edition
1. Euchromatin
the ‘usual ‘ form
contains (potentially) active genes
2. Heterochromatin
more condensed form
additional proteins (HP1)
constitutive
no genes
feature of all cells
e.g., centromeric, telomeric DNA
facultative
in some cells some of the time
inactive genes

32. Mitotic chromosomes are formed from chromatin in its most condensed form
Figure Chromatin packing. This model shows some of the many levels of chromatin packing postulated to give rise to the highly condensed mitotic chromosome.
Figure A typical mitotic chromosome at metaphase. Each sister chromatid contains one of two identical daughter DNA molecules generated earlier in the cell cycle by DNA replication.

33. Each chromosome has a characteristic banding pattern
Figure The banding patterns of human chromosomes. Chromosomes 1-22 are numbered in approximate order of size. A typical human somatic (non-germ line) cell contains two of each of these chromosomes, plus two sex chromosomestwo X chromosomes in a female, one X and one Y chromosome in a male. The chromosomes used to make these maps were stained at an early stage in mitosis, when the chromosomes are incompletely compacted. The horizontal green line represents the position of the centromere (see Figure 4-22), which appears as a constriction on mitotic chromosomes; the knobs on chromosomes 13, 14, 15, 21, and 22 indicate the positions of genes that code for the large ribosomal RNAs (discussed in Chapter 6). These patterns are obtained by staining chromosomes with Giemsa stain, and they can be observed under the light microscope. (Adapted from U. Franke, Cytogenet. Cell Genet. 31:2432, 1981.)

34. Each eukaryotic chromosome has one centromere, two telomeres and many origins of replication (ori)
Molecular Biology of the Gene, 5th Edition
Figure 7-6. Centromeres, origins of replication, and telomeres are required for eukaryotic chromosome maintenance. Each eukaryotic chromosome includes two telomeres, one centromere, and many origins of replication. Telomeres are located at each end of each chromosome. Unlike telomeres, the single centromere found on each chromosome is not in a defined position. Some centromeres are near the middle of the chromosome and others are closer to a telomere. Origins of replication are located throughout the length of each chromosome (approximately every 30 kb in the budding yeast S. cerevisiae)