Chapter 12 Lecture Outline Copyright © 2016 McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education.

12.1 Organization of Functional Sites Along Bacterial Chromosomes The organization of functional sites along bacterial chromosomes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Chromosomes are the structures that contain the genetic material They are complexes of DNA and proteins The genome comprises all the genetic material that an organism possesses In bacteria, it is typically a single circular chromosome In eukaryotes, it refers to one complete set of nuclear chromosomes Note: Eukaryotes also possess a mitochondrial genome Plants also have a chloroplast genome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The DNA molecule does that through its base sequence The main function of the genetic material is to store information required to produce an organism The DNA molecule does that through its base sequence DNA sequences are necessary for Synthesis of RNA and cellular proteins Replication of chromosomes Proper segregation of chromosomes Compaction of chromosomes (so they fit in the cell) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Bacterial Chromosomes Usually a circular molecule that is a few million nucleotides long Escherichia coli, ~ 4.6 million base pairs Haemophilus influenzae, ~ 1.8 million base pairs A typical bacterial chromosome contains a few thousand different genes Protein-encoding genes (structural genes) account for the majority of bacterial DNA The nontranscribed DNA segments between genes are called intergenic regions Play roles in DNA folding, replication, gene regulation, and genetic recombination Origin of replication Genes Intergenic regions Repetitive sequences

12.2 Structure of Bacterial Chromosomes The two processes that make a bacterial chromosome more compact Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Structure of Bacterial Chromosomes The bacterial chromosome is found in a region of the cell called the nucleoid The nucleoid is not surrounded by a membrane So the DNA is in direct contact with the cytoplasm To fit within the bacterial cell, the chromosomal DNA must be compacted about a 1000-fold Proteins organize the DNA into loops Number of loops varies according to chromosome size and the species E. coli has 50-100 with 40,000 to 80,000 bp of DNA in each DNA supercoiling acts to further compact the chromosome, using twisting forces (a) Circular chromosomal DNA Formation of loop domains (b) Looped chromosomal DNA with associated proteins Loop domains DNA- binding proteins The looped structure compacts the chromosome about 10-fold (c) Looped and supercoiled DNA Supercoiling Supercoiling within loops creates a more compact chromosome

12.3 Organization of Functional Sites Along Eukaryotic Chromosomes The organization of functional sites along a eukaryotic chromosome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic Chromosomes Eukaryotic species contain one or more sets of chromosomes Each set is composed of several different linear chromosomes The total amount of DNA in eukaryotic species is typically greater than that in bacterial cells Chromosomes in eukaryotes are located in the nucleus Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Organization of Eukaryotic Chromosomes A eukaryotic chromosome contains a long, linear DNA molecule Three types of DNA sequences are required for chromosomal replication and segregation Origins of replication Centromeres Telomeres Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Origins of replication – several per chromosome Centromere – constricted region of the chromosome, which has a role in chromosome segregation during mitosis and meiosis Kinetochore proteins – group of proteins that link centromere to spindle apparatus Telomere – at ends, prevent translocations and are necessary for maintenance of chromosome length Eukaryotic chromosomes contain many origins of replication approximately 100,000 bp apart Required for proper segregation during mitosis and meiosis Prevent chromosome sticky ends and shortening Centromere Kinetochore proteins Origin of replication Telomere

In lower eukaryotes (such as yeast) Genes are relatively small Genes are located between the centromeric and telomeric regions along the entire chromosome A single chromosome usually has a few hundred to several thousand genes In lower eukaryotes (such as yeast) Genes are relatively small They contain primarily the sequences encoding the polypeptides i.e., Very few introns are present In higher eukaryotes (such as mammals) Genes are long They tend to have many introns intron lengths range from less than 100 to more than 10,000 bp Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12.4 Sizes of Eukaryotic Genomes and Repetitive Sequences Variation in size of eukaryotic genomes Repetitive sequence and how they affect genome sizes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Sizes of Eukaryotic Genomes Genome sizes vary greatly between species Variation is not necessarily related to complexity Example: Salamander species There is a two-fold difference in the size of the genome in two closely related salamander species Size variation is not because of extra genes It is due to accumulation of repetitive DNA sequences Sequences that do not encode proteins Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Has a genome that is more than twice as large as that of P. richmondi Fungi Vascular plants Insects Mollusks © Simpson’s Nature Photography Fishes (b) Plethodon richmondi Salamanders Amphibians Reptiles Birds Mammals 106 107 108 109 1010 1011 1012 (a) Genome sizes (nucleotide base pairs per haploid genome) © William Leonard (c) Plethodon Iarselli Has a genome that is more than twice as large as that of P. richmondi

Unique and Repetitive Sequences Sequence complexity refers to the number of times a particular base sequence appears in the genome There are three main types of DNA sequences Unique or non-repetitive Moderately repetitive Highly repetitive Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Unique or non-repetitive sequences Found once or a few times in the genome Includes structural genes as well as introns and other noncoding DNA In humans, make up roughly 41% of the genome Percentage in the human genome Classes of DNA sequences 60 40 20 100 Regions of genes that encode proteins (exons) 2% 24% 15% 59% 80 Introns and other parts of genes Unique noncoding DNA Repetitive Unique sequences

Moderately repetitive Found a few hundred to a few thousand times Includes Genes for rRNA and histones Origins of replication Transposable elements Discussed in detail in Chapter 21 Highly repetitive Found tens of thousands to millions of times Each copy is relatively short (a few nucleotides to several hundred in length) Some sequences are interspersed throughout the genome Example: Alu family in humans Others are clustered together in tandem arrays Example: AATAT and AATATAT in Drosophila Commonly found in the centromeric regions

12.5 Structure of Eukaryotic Chromosomes in Nondividing Cells Definition of chromatin The structures of nucleosomes, the 30-nm fiber, and radial loop domains Noll’s results and how they support the beads-on-a-string model Description of a chromosome territory Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic Chromatin Compaction If stretched end to end, a single set of human chromosomes would be over 1 meter long! Yet the cell’s nucleus is only 2 to 4 mm in diameter Therefore, DNA must be tightly compacted to fit The compaction of linear DNA in eukaryotic chromosomes involves interactions between DNA and various proteins In eukaryotes, this DNA-protein complex is known as chromatin Changes in the proteins affect the degree of chromatin compaction during the life of the cell Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Diameter of the nucleosome Nucleosomes The repeating structural unit within eukaryotic chromatin is the nucleosome A nucleosome is composed of double-stranded DNA wrapped around an octamer of histone proteins An octamer is composed two copies each of four different histones H2A, H2B, H3, and H4 146 bp of DNA make 1.65 superhelical turns around the octamer Varies in length between 20 to 100 bp, depending on species and cell type Diameter of the nucleosome

Histone proteins are basic They contain many positively-charged amino acids Lysine and arginine These bind to the negatively charged phosphates along the DNA backbone via electrostatic interactions and hydrogen bonds Histone proteins have a globular domain and a flexible, charged amino terminus or ‘tail’ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Play a role in the organization and compaction of the chromosome There are five types of histones H2A, H2B, H3 and H4 are the core histones Two of each make up the octamer H1 is the linker histone Binds to linker DNA Also binds to nucleosomes But not as tightly as are the core histones Nonhistone proteins also bind the linker region, aid in chromosome compaction and affect gene expression (c) Nucleosomes showing linker histones and nonhistone proteins Histone octamer Histone H1 Nonhistone proteins Linker DNA Play a role in the organization and compaction of the chromosome

Nucleosomes Join to Form a 30-nm Fiber Nucleosomes associate with each other to form a more compact structure – the 30-nm fiber Shortens the total length of DNA 7-fold Structure has been difficult to determine Two models have been proposed Solenoid model Zigzag model (b) Solenoid model (c) Zigzag model 30 nm Core histone proteins Regular, spiral configuration containing six nucleosomes per turn Irregular configuration where nucleosomes have little face-to-face contact

Further Compaction of the Chromosome Together the nucleosomes and 30-nm fiber shorten the DNA about 50-fold A third level of compaction involves interaction between the 30-nm fiber and the nuclear matrix The nuclear matrix Composed of two parts Nuclear lamina — fibers lining the inner membrane Internal nuclear matrix — connects to lamina, fills nucleus interior Compacts DNA into radial loop domains Protein bound to internal nuclear matrix filament Internal nuclear matrix Nuclear lamina (a) Proteins that form the nuclear matrix Outer membrane Inner pore

Radial Loop Domains Matrix-attachment regions (MARs) also known as Scaffold-attachment regions (SARs) are DNA sequences interspersed in the genome They anchor to the nuclear matrix, forming radial loops (d) Radial loop bound to a nuclear matrix fiber MAR Radial loop 30-nm fiber Protein Gene Matrix-attachment regions (MARs) or Scaffold-attachment regions (SARs) MARs are anchored to the nuclear matrix, thus creating radial loops 25,000 to 200,000 bp

Chromosome Territories The nuclear matrix organizes the chromosomes within the nucleus In interphase, each chromosome is located in a distinct chromosome territory These are visible when chromosomes are fluorescently labeled

Heterochromatin vs. Euchromatin The compaction of interphase chromosomes is not completely uniform Euchromatin Less condensed regions of chromosomes Transcriptionally active Regions where 30-nm fiber forms radial loop domains Heterochromatin Tightly compacted regions of chromosomes Transcriptionally inactive (in general) Radial loop domains compacted even further Telomere Euchromatin (30-nm fiber anchored in radial loops) Heterochromatin (greater compaction of the radial loops) Centromere

12.6 Structure of Eukaryotic Chromosomes During Cell Division The levels of compaction that lead to a metaphase chromosome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Metaphase Chromosomes As cells enter M phase, the level of compaction changes dramatically By the end of prophase, sister chromatids are entirely heterochromatic Two parallel chromatids have an overall diameter of 1,400 nm These highly condensed metaphase chromosomes undergo little gene transcription

Compaction level in euchromatin Compaction level in heterochromatin In metaphase chromosomes, the radial loops are highly compacted and stay anchored to a scaffold Histones are needed for the compaction of radial loops Compaction level in euchromatin Compaction level in heterochromatin