Rosalind Morris

Engineering the Genome

• DNA transformation has removed most of the barriers that limit the introduction of specific genes from one organism to another. • The use of Agrobacteria and modified Ti plasmids to transform dicotyledons, and the use of microinjection to transfect the nuclei of animal cells, are being superseded by particle-bombardment biolistic procedures, developed from the need to transform monocotyle-donous crops. • Chimeric-gene constructs combine the gene to be transformed with regulator and promoter sequences suitable for the host, in order to improve the stability or rate of formation of the gene product in the transformed host. • Through DNA transformation, certain plants and animals can be regarded as factories for the production of novel gene products. • Transformation by antisense genes can block the formation of product from normal sense genes, if necessary in a specific tissue; the insertion of multiple copies of sense genes can have the same effect. • Successfully transformed organisms must transmit introduced genes in a Mendelian manner.

Variable Structure and Folding of DNA

• The three-dimensional, double-helix structure of DNA has numerous variations depending on the sequence of internal base pairs and the presence of interacting external molecules. • Methylation of DNA is a chemical modification of the cytosine and adenosine residues that results in changes in the control of gene expression and DNA imprinting. • Proteins interacting with DNA to control gene expression have characteristic structures and distort the double helix upon binding. • The folding of DNA into chromosomes involves the formation of nucleosomes, which consist of a core of four different histones with DNA wrapped around the outside. • Further folding involves the ordering of nucleosomes into fibers and chromatin domains by the formation of loops, which are attached at their bases to a nuclear scaffold. • Single chromosomes tend to form domains within the nucleus and have nuclear-membrane attachment sites.

Microscopes: Basic Tools for Cytogenetics

• The standard light microscope has many variations based on manipulating the path of light through the optical system. • The wavelength of light is a major determinant of resolution in the microscope, and the shorter “wavelength” of electrons provides greater magnification with the electron microscope. • Fluorescence provides a versatile basis for tracing specific features of chromosomes. • Computer capture of microscopic images has enhanced the analytical power of microscopes.

Replication of Protosomes and Chromosomes

• The replication of DNA is semiconservative, involving a single origin of replication in prokaryotes and multiple origins of replication in eukaryotes. • Synthesis of new DNA occurs by copying an existing template through the addition of 5′-nucleoside monophosphate units to the 3′OH moiety of a primer sequence. • Many proteins are involved in the replication process, and the enzymatic activities are the same in prokaryotes and eukaryotes. • The high fidelity of DNA replication is the result of several different postreplicative editing activities that remove errors. • Any single DNA segment is usually replicated only once per cell cycle, and the assembly of chromatin after DNA replication in eukaryotes is a critical period for competition between transcription-control factors and histones. • Developmentally regulated deviations from the “once only per cell cycle” rule for DNA replication can lead to amplification of regions of the genome.

A Historical Perspective on Chromosome Structure, Function, and Behavior

• The pre-1900 period established the cell theory, the concept of cell lineage, the microscopic structure of chromosomes, mitotic and meiotic cell divisions, and the existence of genes in nuclear chromatin. • Studies during the 1900–1950 period recognized the linear order of genes in chromosomes, established the “one gene-one protein” concept, used karyotyping to investigate genetic and evolutionary relatedness, related changes in chromosome structure to mutations, provided evidence for transposable elements, and proved that DNA was the chemical component of genes. • The 1950–1996 period established the physical structure of chromosomes, including the three-dimensional configuration of DNA and the histone proteins bound to it, the banding of whole chromosomes, and the distribution of specific DNA sequences. • The universality of the genetic code was established in this period, as were techniques for transforming bacteria, plants, and animals, and ever-increasing uses for the polymerase chain reaction. • Worldwide DNA sequence databases and associated computer technology for utilizing the information were devised, culminating, for the moment, in the sequencing of the entire yeast genome.

Chromosomes in the Mitotic Cell Cycle

• The cell cycle is comprised of a chromosome-replication cycle occurring in concert with a cytoplasm-replication cycle. • The centrosomes determine the polarity of cell division, and centromeres (either well-defined chromosome regions or diffuse) play an active role in the movement of chromosomes to the poles. • The cell cycle is regulated by a series of protein phosphorylation and dephosphorylation reactions controlled by cell-division-cycle (cdc) genes. • Mutations in the cdc genes lead to uncontrolled cell growth and are the primary cause of many types of cancer.

Organization of DNA Sequences in Protosomes and Chromosomes

• The amounts of nuclear DNA vary greatly between organisms even though (80–100) × 106 base pairs of DNA are theoretically sufficient to define a basic set of genes necessary for eukaryotic life-forms. • Under suitable experimental conditions, denatured DNA spontaneously reforms a double helix, based on A-T and G-C base pairing. • Repetitive DNA sequences are a major source of variation in DNA amount and contribute to modulating gene activity. • Many repetitive sequences are dispersed throughout the genome and were transposable or retrotransposable elements at some stage in their evolutionary history. • Some regions of DNA that affect the expression of a gene can be thousands of base pairs away from the respective gene.

The Cytogenetic Analysis of Haploids

• Some organisms can survive with half the usual somatic chromosome number. • The smaller size of haploids is due to the reduction in the normal gene dosage of somatic cells. • Haploids arise spontaneously from irregularities in sexual reproduction and are induced by various methods such as chromosome elimination in wide crosses or in vitro anther culture. • Haploids are highly sterile because of the irregular meiotic behavior of unpaired chromosomes. • Doubled-haploid plants are genetically homozygous and provide an important basis for producing new varieties with greater efficiency.

Exploring Chromosomes by In Situ Biochemical Reactions

• Reactions carried out on chromosomes in situ involve a primary reaction that defines the specificity of the reaction, and a secondary reaction that provides the means for detecting the product of the primary reaction. • The chemicals used in the preparation of chromosomes can determine the specificity of the primary reaction. • Antibodies that detect specific features of chromosomes often provide the basis for a primary reaction. • The hybridization of a nucleic-acid probe to its complementary sequence in chromosomal DNA is widely used in primary reactions to determine the sequence organization in chromosomal DNA. • The polymerase chain reaction (PCR) provides a highly sensitive primary reaction for characterizing chromosome structure. • Secondary reactions utilize enzymes that cause the precipitation of a dye at the site of the primary reaction, or bind a fluorescent-labeled antibody to a component of the primary reaction.

Multiples of Basic Chromosome Numbers—Polyploidy

• Polyploids have more than two basic chromosome sets and they occur mainly in plants. • A major cause of spontaneous polyploidy in plants is unreduced gametes resulting from meiotic irregularities. • Doubling chromosome numbers in somatic cells by means of colchicine is the universal method for inducing polyploidy in plants. • The phenotypic consequences of polyploidy result from increased dosages of genes in each cell of all or some of the tissues of an organism, and they vary depending on whether the organism is autopolyploid or allopolyploid. • The diploidlike pairing in many natural allopolyploids is due to the presence of genes that prevent pairing between partially homologous chromosomes. • Gene segregations in polyploids depend on the relationships between the constituent genomes, and the meiotic behavior of the homologous or partially homologous chromosomes. • Genome analysis is based on quantitative analyses of meiotic pairing, genetic compensation of substituted chromosomes, and homoeo-logous relationships between genomes.