Understanding the Human Genome

 Student project / Johns Hopkins Medical Institutions, Division of Biomedical Information Sciences (undated)

Fig. 1. The Human Genome at Four Levels of Detail

Apart from reproductive cells (gametes) and mature red blood cells, every cell in the human body contains 23 pairs of chromosomes, each a packet of compressed and entwined DNA (1, 2). Each strand of DNA consists of repeating nucleotide units composed of a phosphate group, a sugar (deoxyribose), and a base (guanine, cytosine, thymine, or adenine) (3). Ordinarily, DNA takes the form of a highly regular double- stranded helix, the strands of which are linked by hydrogen bonds between guanine and cytosine and between thymine and adenine. Each such linkage is a base pair (bp); some 3 billion bp constitute the human genome. The specificity of these base- pair linkages underlies the mechanism of DNA replication illustrated here. Each strand of the double helix serves as a template for the synthesis of a new strand; the nucleotide sequence (i.e., linear order of bases) of each strand is strictly determined. Each new double helix is a twin, an exact replica, of its parent. (Figure and caption text provided by the LBL Human Genome Center.)

Fig. 2. DNA Structure

The four nitrogenous bases of DNA are arranged along the sugar- phosphate backbone in a particular order (the DNA sequence), encoding all genetic instructions for an organism. Adenine (A) pairs with thymine (T), while cytosine (C) pairs with guanine (G). The two DNA strands are held together by weak bonds between the bases.

A gene is a segment of a DNA molecule (ranging from fewer than 1 thousand bases to several million), located in a particular position on a specific chromosome, whose base sequence contains the information necessary for protein synthesis.

Fig. 3. Comparison of Largest Known DNA Sequence with Approximate Chromosome and Genome Sizes of Model Organisms and Humans

A major focus of the Human Genome Project is the development of sequencing schemes that are faster and more economical.

Comparative Sequence Sizes                (Bases)

(yeast chromosome 3)                   350 Thousand

Escherichia coli (bacterium) genome    4.6 Million

Largest yeast chromosome now mapped    5.8 Million

Entire yeast genome                     15 Million

Smallest human chromosome (Y)           50 Million

Largest human chromosome (1)           250 Million

Entire human genome                      3 Billion

Fig. 4. DNA Replication.

During replication the DNA molecule unwinds, with each single strand becoming a template for synthesis of a new, complementary strand. Each daughter molecule, consisting of one old and one new DNA strand, is an exact copy of the parent molecule. [Source: adapted from Mapping Our GenesThe Genome Projects: How Big, How Fast? U.S. Congress, Office of Technology Assessment, OTA- BA- 373 (Washington, D.C.: U.S. Government Printing Office, 1988).]

Fig. 5. Gene Expression.

When genes are expressed, the genetic information (base sequence) on DNA is first transcribed (copied) to a molecule of messenger RNA in a process similar to DNA replication. The mRNA molecules then leave the cell nucleus and enter the cytoplasm, where triplets of bases (codons) forming the genetic code specify the particular amino acids that make up an individual protein. This process, called translation, is accomplished by ribosomes (cellular components composed of proteins and another class of RNA) that read the genetic code from the mRNA, and transfer RNAs (tRNAs) that transport amino acids to the ribosomes for attachment to the growing protein. (Source: see Fig. 4.)

Fig. 6. Karyotype.

Microscopic examination of chromosome size and banding patterns allows medical laboratories to identify and arrange each of the 24 different chromosomes (22 pairs of autosomes and one pair of sex chromosomes) into a karyotype, which then serves as a tool in the diagnosis of genetic diseases. The extra copy of chromosome 21 in this karyotype identifies this individual as having Down's syndrome.

Fig. 7. Assignment of Genes to Specific Chromosomes. The number of genes assigned (mapped) to specific chromosomes has greatly increased since the first autosomal (i.e., not on the X or Y chromosome) marker was mapped in 1968. Most of these genes have been mapped to specific bands on chromosomes. The acceleration of chromosome assignments is due to (1) a combination of improved and new techniques in chromosome sorting and band analysis, (2) data from family studies, and (3) the introduction of recombinant DNA technology. [Source: adapted from Victor A. McKusick, Current Trends in Mapping Human Genes, The FASEB Journal 5(1), 12 (1991).]

HUMAN GENOME PROJECT GOALS

Resolution

• Complete a detailed human genetic map 2 Mb

• Complete a physical map 0.1 Mb

• Acquire the genome as clones 5 kb

• Determine the complete sequence 1 bp

• Find all the genes

With the data generated by the project, investigators will determine the functions of the genes and develop tools for biological and medical applications.

Fig. 8. Constructing a Genetic Linkage Map.

Genetic linkage maps of each chromosome are made by determining how frequently two markers are passed together from parent to child. Because genetic material is sometimes exchanged during the production of sperm and egg cells, groups of traits (or markers) originally together on one chromosome may not be inherited together. Closely linked markers are less likely to be separated by spontaneous chromosome rearrangements. In this diagram, the vertical lines represent chromosome 4 pairs for each individual in a family. The father has two traits that can be detected in any child who inherits them: a short known DNA sequence used as a genetic marker (M) and Huntingtons disease (HD). The fact that one child received only a single trait (M) from that particular chromosome indicates that the fathers genetic material recombined during the process of sperm production. The frequency of this event helps determine the distance between the two DNA sequences on a genetic map .

Fig. 9. Physical Mapping Strategies.

Top-down physical mapping (a) produces maps with few gaps, but map resolution may not allow location of specific genes. Bottom- up strategies (b) generate extremely detailed maps of small areas but leave many gaps. A combination of both approaches is being used. [Source: Adapted from P. R. Billings et al., New Techniques for Physical Mapping of the Human Genome, The FASEB Journal 5(1), 29 (1991).] Note: Figure 9 was missing from the original file)

Fig. 10. Types of Genome Maps.

At the coarsest resolution, the genetic map measures recombination frequency between linked markers (genes or polymorphisms). At the next resolution level, restriction fragments of 1 to 2 Mb can be separated and mapped. Ordered libraries of cosmids and YACs have insert sizes from 40 to 400 kb. The base sequence is the ultimate physical map. Chromosomal mapping (not shown) locates genetic sites in relation to bands on chromosomes (estimated resolution of 5_Mb); new in situ hybridization techniques can place loci 100 kb apart. This direct strategy links the other four mapping approaches. [Source: see Fig. 9.Note: Figure 9 is missing]

Fig. 11. Constructing Clones for Sequencing.

Cloned DNA molecules must be made progressively smaller and the fragments subcloned into new vectors to obtain fragments small enough for use with current sequencing technology. Sequencing results are compiled to provide longer stretches of sequence across a chromosome. (Source: adapted from David A. Micklos and Greg A. Freyer, DNA Science, A First Course in Recombinant DNA Technology, Burlington, N.C.: Carolina Biological Supply Company, 1990.)

DNA Amplification: Cloning

(a) Cloning DNA in Plasmids

By fragmenting DNA of any origin (human, animal, or plant) and inserting it in the DNA of rapidly reproducing foreign cells, billions of copies of a single gene or DNA segment can be produced in a very short time. DNA to be cloned is inserted into a plasmid (a small, self- replicating circular molecule of DNA) that is separate from chromosomal DNA. When the recombinant plasmid is introduced into bacteria, the newly inserted segment will be replicated along with the rest of the plasmid.

(b) Constructing an Overlapping Clone Library.

A collection of clones of chromosomal DNA, called a library, has no obvious order indicating the original positions of the cloned pieces on the uncut chromosome. To establish that two particular clones are adjacent to each other in the genome, libraries of clones containing partly overlapping regions must be constructed. These clone libraries are ordered by dividing the inserts into smaller fragments and determining which clones share common DNA sequences.

Fig. 12. DNA Sequencing.

Dideoxy sequencing (also called chain- termination or Sanger method) uses an enzymatic procedure to synthesize DNA chains of varying lengths, stopping DNA replication at one of the four bases and then determining the resulting fragment lengths. Each sequencing reaction tube (T, C, G, and A) in the diagram contains

• a DNA template, a primer sequence, and a DNA polymerase to initiate synthesis of a new strand of DNA at the point where the primer is hybridized to the template;

• the four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) to extend the DNA strand;

• one labeled deoxynucleotide triphosphate (using a radioactive element or dye); and

• one dideoxynucleotide triphosphate, which terminates the growing chain wherever it is incorporated. Tube A has didATP, tube C has didCTP, etc.

For example, in the A reaction tube the ratio of the dATP to didATP is adjusted so that each tube will have a collection of DNA fragments with a didATP incorporated for each adenine position on the template DNA fragments. The fragments of varying length are then separated by electrophoresis (1) and the positions of the nucleotides analyzed to determine sequence. The fragments are separated on the basis of size, with the shorter fragments moving faster and appearing at the bottom of the gel. Sequence is read from bottom to top (2). (Source: see Fig. 11.)

Fig. 13. Cloning a Disease Gene by Chromosome Walking.

After a marker is linked to within 1_cM of a disease gene, chromosome walking can be used to clone the disease gene itself. A probe is first constructed from a genomic fragment identified from a library as being the closest linked marker to the gene. A restriction fragment isolated from the end of the clone near the disease locus is used to reprobe the genomic library for an overlapping clone. This process is repeated several times to walk across the chromosome and reach the flanking marker on the other side of the disease- gene locus. (Source: see Fig. 11.)

HUMAN GENETIC DIVERSITY:

The Ultimate Human Genetic Database

• Any two individuals differ in about 3 x 106 bases (0.1%).

• The population is now about 5 x 109.

• A catalog of all sequence differences would require 15 x 1015 entries.

• This catalog may be needed to find the rarest or most complex disease genes.

Fig. 14. Magnitude of Genome Data.

If the DNA sequence of the human genome were compiled in books, the equivalent of 200 volumes the size of a Manhattan telephone book (at 1000 pages each) would be needed to hold it all. New data- analysis tools will be needed for understanding the information from genome maps and sequences.

Fig. 15. Understanding Gene Function.

Understanding how genes function will require analyses of the 3- D structures of the proteins for which the genes code.







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