HAPLOID GENOME SIZES (DNA PER HAPLOID CELL)

Size range Example species Ex. Size BACTERIA 1-10 Mb E. coli: 4.639 MbFUNGI 10-40 Mb S. cerevisiae 13 MbINSECTS 100-5000 Mb D. melanogaster 165 MbBIRDS 1000-1500 Mb Chicken 1300 Mb FLOWERING 100 to Arabidopsis 120 MbPLANTS 100,000 Mb Corn 2500 MbMAMMALS 3000-4000 Mb Human 3000 Mb

1 Mb = 1 million base pairs. (Probably the number of essential genes does not differ greatly among various multicellular organisms. Most estimates are that humans have about 40,000 genes.)

KEY DIFFERENCES INPROKARYOTIC VS. EUKARYOTIC

GENOMES:

SIZE: Haploid human genome ~700 X E. coli genome

GENE NO.: Human genome has ~10 X the genes of E. coli

MULTIPLE LINEAR CHROMOSOMES

DIPLOID (2n) FOR MOST OF LIFE CYCLE

DIFFERENT SEXUAL CYCLE = MEIOSIS

2n chromosomes segregate during meiosis (each pair segregates independently of the other pairs)to generate haploid (n) germ cells. Germ cells unite to form 2n zygote/organism. In addition to the genetic reassortment of different chromosome pairs, additional reassortment arises from recombination between the two chromosomes of each pair

DIFFERENT PHENOTYPES OF MOST INTEREST

(e.g., complex developmental, behavioral, etc., traits for which there often is no obvious biochemical explanation via a known enzyme or gene.

EUKARYOTIC GENOMES CONSIST OF VARIABLE AMOUNTS OF 3 CLASSES OF DNA

A. Single copy or unique DNA sequences: present once per haploid genome. B. Moderately repetitive DNA: present in 10-1000 copies per haploid genomeC. Highly repetitive DNA: present in thousands of copies per haploid genome;

GENOME MAPS: Genome maps allow us to understand the specific arrangment of genes and other sequences on each chromosome of a given species. This is important when we wish to relate one gene or landmark to another in the genome. Genome maps are of 4 major types.

A. GENETIC: measures distances between genes by the RECOMBINATION FREQUENCY between Mendelian genetic markers

B. CYTOLOGICAL (or cytogenetic): measures distances between VISIBLE BANDS OR STAINED REGIONS found in chromosome KARYOTYPES

C. RADIATION HYBRID: measures distances between markers according to the likelihood of a radiation-induced chromosome break between them

D. PHYSICAL: measures distances between genetic elements in terms of the LENGTH OF DNA between them.

GENETIC LINKAGE MAPPING

A. POLYMORPHISM: In order to map genes, one must have at least two different alleles for any given gene and the two alleles must give rise to an observable phenotype. This difference is called POLYMORPHISM. Three of these polymorphic assays will be discussed further:

B. RFLP, Restriction Fragment Length Polymorphism

C. STSs (SEQUENCE TAGGED SITE) A short region of DNA whose sequence is known, so that it can be amplified by PCR; may contain sequence polymorphisms.

One particularly useful type of STS is the microsatellite marker. A microsatellite is an STS which contains a tandem repeat of a very simple DNA sequence, e.g., (CA)n. Because errors are made in replicating such sequences the “n” often varies from one individual to another (i.e., it is polymorphic.)

D. SNPs (Single Nucleotide Polymorphisms). A single nucleotide difference between the sequences of two homologous chromosomes (for example, the homologous chromosome 1 copies that you received, one from your mother and one from your father). Most human haploid genomes differ by about 1-3 million SNPs from each other. There are a variety of mechanisms used to identify SNPs. The disadvantage of SNPs is that they are not as polymorphic as microsatellite sequences.

PHYSICAL MAPPING OF EUKARYOTIC CHROMOSOMES REQUIRES THAT WE BE ABLE TO HANDLE VERY LARGE DNAs (~1 Mb).

A. Sizing and Separating Large DNA Fragments: Large DNA fragments (up to 10 Mb) can be separated on special gels in which the direction of the electric field is altered in pulses (PULSED FIELD GEL ELECTROPHORESIS).

B. Large Scale Restriction Maps:

C. Cloning Large DNA: Large DNA fragments can be cloned in special vectors, such as YEAST ARTIFICIAL CHROMOSOME (YAC) AND BACTERIAL ART. CHROMOSOME (BAC) VECTORS.

How to correlate genetic, cytological and physical maps? A Cloned Gene or STS is the key. (Remember, any time you have an STS, you can always use PCR to make lots of that DNA.)

A. Physical maps are often restriction maps produced by overlapping cloned DNA fragments.

B. Genetic maps are produced using RFLPs deduced from Southern blots using cloned DNA as the probe or STSs using DNA sequence information derived by sequencing a cloned DNA fragment.

C. Cytological maps are derived using in situ hybridization, again using a cloned DNA as the hybridization probe.

XII. The utimate physical map is the complete sequence of the genome. (pp. 152-156)

GENOME SIZE (Mb) PHYSICAL MAP COMPLETE SEQUENCE

phage DNA 0.05 ca. 1970 1982EBV 0.2 ca. 1980 1984H. influenzae 1.8 1995 July, 1995E. coli 4.7 1987 Feb. 1997S. cerevisiae 13 ca. 1990 total 4/24/96C. elegans 80 near, 1993 Dec. 1998D. melanogaster 140 est. 1999

2/2000Arabidopsis 120 1992-3 est. 2000

12/2000Human 3000 1st gen., Dec. 1993 est. 2003-5

draft: Feb. 2001finished, est. 2003