eChapters

General Effect of Mutation

Gene mutations are molecular changes in the DNA sequence of a gene

Gene mutations can alter the coding sequence within a gene

Gene mutations are also given names that describe how they affect the wild-type genotype and phenotype

Gene mutations can occur outside of the coding sequence and still influence gene expression

DNA sequences known as trinucleotide repeats may cause mutation

Changes in chromosome structure can affect the expression of a gene

Mutations can occur in germ line or somatic cells

The Structure of Proteins

Proteins are composed of building blocks called amino acids. A string of amino acids is called a polypeptide chain. Once such a chain has folded into its working three-dimensional shape, it is a protein. Though there are tens of thousands of different proteins, all of them are put together from a starting set of 20 amino acids. It is the order in which the amino acids are linked in a polypeptide chain that determines which protein will be produced.

The Genetic Basis for Protein Synthesis

Archibald Garrod proposed that some genes code for the production of a single enzyme

Beadle and Tatum’s experiments with Neurospora led them to propose the one gene-one enzyme hypothesis

During translation, the genetic code within mRNA is used to make a polypeptide with a specific amino acid sequence

Exceptions to the genetic code are known to occur, which include the incorporation of selenocysteine and pyrrolysine into polypeptides

Synthetic RNA helped to decipher the genetic code

The use of RNA copolymers and the triplet binding assay also helped to crack the genetic code

A polypeptide chain has directionality from its amino terminal to its carboxyl terminal end

The amino acid sequences of polypeptides determine the structure and function of proteins

Cellular proteins are primarily responsible for the characteristics of living cells and an organism’s traits

Overview of Transcription

Gene expression requires base sequences that perform different functional roles

The three stages of transcription are initiation, elongation, and termination

RNA transcripts have different functions Transcription in Bacteria

A promoter is a short sequence of DNA that is necessary to initiate transcription

Bacterial transcription is initiated when RNA polymerase holoenzyme binds at a promoter sequence

The RNA transcript is synthesized during the elongation stage

Transcription is terminated by either an RNA-binding protein or an intrinsic terminator

Identification of DNA as the Genetic Material

Experiments with pneumococcus suggested that DNA is the genetic material

Hershey and Chase provided evidence that the genetic material injected into the bacterial cytoplasm is T2 phage DNA

RNA functions as the genetic material in some viruses Work Before Watson and Crick

Nucleotides are the building blocks of nucleic acids

Nucleotides are linked together to form a strand

In 1869, Miescher first isolated DNA and named it “nuclein.”

In 1902, Archibald Garrod linked heredity to enzyme (protein) defects. In 1909 he published a book entitled “Inborn Errors of Metabolism.”

In 1928, Griffith identified a “transforming factor” that transmitted infectiousness in bacteria.

In 1944, Avery, MacLeod and McCarty showed that the transforming factor was DNA)

In 1950, Hershey and Chase, using their famous “blender” experiment, confirmed that DNA, not protein, is the genetic material.

The work of chemist Phoebus Levene showed that DNA includes deoxyribose, nitrogenous bases, and phosphates, in equal proportions.

Chargaff s data showed that the number of purines equals the number of pyrimidines.

Using Rosalind Franklin’s X-ray diffraction patterns, Watson and Crick deduced that DNA is a double helix

Variation in Chromosome Structure

Natural variation exists in chromosome structure

Changes in chromosome structure include deletions, duplications, inversions, and translocations

The loss of genetic material in a deletion tends to be detrimental to an organism

Duplications tend to be less harmful than deletions

Duplications provide additional material for gene evolution, sometimes leading to the formation of gene families

Copy number variation is relatively common among members of the same species

Comparative genomic hybridization is used to detect chromosome deletions and duplications

Inversions often occur without phenotypic consequences

Inversion heterozygotes may produce abnormal chromosomes due to crossing over

Translocations involve exchanges between different chromosomes

Individuals with reciprocal translocations may produce abnormal gametes due to the segregation of chromosomes

Portrait of a Chromosome

• Cytogenetics is the study of chromosome abnormalities and associated effects on health or other traits.
• Excess or deficient genetic material can cause syndromes or end prenatal development.
• Chromosomes consist of highly coiled DNA, RNA, histones, and nonhistone proteins.

Telomeres and Centromeres are Essential

Telomeres consist of repeat sequences and protect chromosome tips.

A centromere is a constricted site where spindle fibers attach during cell division.

Centromeres are regions of repeated DNA bound to centromere-associated proteins.

Sex Chromosomes

Sex can be considered at chromosomal, gonadal, phenotypic, and gender identity levels.

Human females are homogametic (XX) and males are heterogametic (XY).

The Y chromosome contains few identified genes, while the larger X chromosome contains several thousand genes. Pseudoautosomal regions at both tips of the Y chromosome contain genes that have counterparts on the X chromosome.

The male determining gene, SRY, has been found on the Y chromosome.

Genetic Transfer and Mapping in Bacteria a

Bacteria can transfer genetic material during conjugation

Hfr strains contain an F factor integrated into the bacterial chromosome

Hfr strains can transfer a portion of the bacterial chromosome to recipient cells

Conjugation experiments can map genes along the E. coli chromosome

A genetic map of the E. coli chromosome has been obtained from many conjugation studies

Bacteria may contain different types of plasmids

Bacteriophages transfer genetic material from one bacterial cell to another via transduction

Cotransduction can be used to map genes that are within 2 minutes of each other

Bacteria can also transfer genetic material by transformation

Bacteria may acquire new genes by horizontal gene transfer

Linkage and Crossing Over

Crossing over may produce recombinant phenotypes.

Bateson and Punnett discovered two traits that did not assort independently.

Morgan provided evidence for the linkage of several X-linked genes and proposed that crossing over between X chromosomes can occur.

A chi square analysis can be used to distinguish between linkage and independent assortment.

Creighton and McClintock correlated crossing over that produced new combinations of alleles with the exchange of homologous chromosomes.

Crossing over occasionally occurs during mitosis. Genetic Mapping in Plants and Animals

The frequency of recombination between two genes can be correlated with their map distance along a chromosome

Alfred Sturtevant used the frequency of crossing over in dihybrid crosses to produce the first genetic map.

Trihybrid crosses can be used to determine the order and distance between linked genes.

Interference can influence the number of double crossovers that occur in a short region.

Genetic Transfer and Mapping in Bacteria

Bacteria can transfer genetic material during conjugation

Hfr strains contain an f factor integrated into the bacterial chromosome

Conjugation experiments can be used to map genes along the E. coli chromosome

A detailed genetic map of the E. coli chromosome has been obtained from many conjugation studies

Bacteriophages can also transfer genetic material from one bacterial cell to another in the process of transduction

Cotransduction can be used to map genes that are within 2 minutes of each other

Bacteria can also transfer genetic material by transformation

Horizontal gene transfer is the transfer of genes among different species

When Gene Expression Appears to Alter Mendelian Ratios Lethal Allele Combinations

Us recessive lethal alleles eliminaters a progeny class. Multiple Alleles

A gene can have more than two alleles, but a diploid individual only has one or two of them.

Different allele combinations can produce different phenotypes and different severities of symptoms.

Different Dominance Relationships

Incomplete dominance of an allele produces a phenotype in the heterozygote that is intermediate between that of either homozygote.

Codominant alleles are both expressed in a heterozygote. Epistasis-When One Gene Affects Expression of Another

In epistasis, one gene masks the effect of another. An example of epistasis in humans is the Bombay phenotype.

The Bombay phenotype results in O type blood for individuals homozygous recessive for the recessive “h” allele.

About Mendel

Born in what is now the Czeck Republic.

Grew up in an agricultural environment.

Became a priest-teacher at a local monastery.

Took courses in botany, physics, and mathematics at the University of Vienna.

Gregor Mendel was the first person to comprehend some of the most basic principles of genetics. He reached these understandings in the mid-nineteenth century; working in what is now the Czech Republic and using as his experimental subjects a species of garden pea, Pisum sativum..

Carried out “hybridization” experiments with the common garden pea.

Mendel’s hypotheses became the laws of inheritance in modern genetics.

Mendel’s work re-discovered by Correns, DeVries, and Tschermak in 1900. Period of modern “Mendelian” genetics begin.

Cells are the Working Units of Life

With the possible exception of viruses, every form of life on Earth either is a cell or composed of cells. All cells come into existence through the activity of other cells.

All Cells are either Prokaryotic or Eukaryotic

All cells can be classified as prokaryotic or eukaryotic. All plants, animals, fungi, and protists are either single eukaryotic cells or are composed of eukaryotic cells. Prokaryotic cells are either bacteria or archaea. Eukaryotic cells have most of their DNA contained in a membrane-lined nucleus, whereas prokaryotic cells do not have a nucleus. Eukaryotic cells also have more specialized structures, called organelles, than do prokaryotic cells. All prokaryotes are single-celled, whereas many eukaryotes are multi-celled.

The Eukaryotic Cell

There are five principal components to the eukaryotic animal cell: the nucleus, other organelles, the cytosol, the cytoskeleton, and the plasma membrane. Organelles are “tiny organs” within the cell that carry out specialized functions, such as energy transfer and material recycling. The cytosol is the fluid in which these organelles are immersed (The cytoplasm is the region of the cell inside the plasma membrane but outside the nucleus.) The cytoskeleton is composed of several groups of proteins that give the cell support and facilitate transportation of cellular elements. The plasma membrane is the chemically active outer boundary of the animal cell. Plant cells have a plasma membrane that has, outside of it, a cell wall.

A Overview

Genetics is a branch of biology concerned with heredity and variation.

Genes are the unit of inheritance and are composed of DNA.

The effects of genes are noticeable at the molecular, cellular, family, and population levels.

Genes are DNA sequences that instruct cells to produce particular proteins, which in turn determine traits.

Genes can exist in more than one form and the variants of a gene arise by mutation.

An allele is a variant of a gene that alters its expression in a detectable way. Alleles can be dominant or recessive.

A polymorphism is a particular sequence of DNA that varies in one percent or more of the population.

Single nucleotide polymorphisms are single base pair sites that differ among individuals.

Chromosomes consist of genes and associated proteins.

The human genome consists of 22 pairs of autosomes and one pair of sex chromosomes.

A karyotype is the photographic catalog of the human chromosomes.

Specialized cells and tissues arise by differentiation from the stem cells of the early embryo.

Pedigree diagrams enable recessive and dominant traits to be followed through multiple generations of a family.

A gene pool is the collection of alleles in a population.

Comparative genomics is leading to a more detailed and subtle understanding of evolutionary relationships among species

After preparing the desired DNA molecule using E.coli, the plasmid can be transferred into Agrobacterium by this method. Efficiency is low (~10³ transformants per mg of DNA) but the method is reliable and very rapid. This method also eliminates much of the plasmid rearrangement that occurs during triparental mating.

Procedure

Grow an Agribacterium strain containing an appropriate helper Ti plasmid in 5 ml of LB medium 16 hours at 28ºC.

Add 2 ml of the culture to 50 ml LB medium in a 250 ml flask and shake (~250 RPM) at 28ºC until an OD₆₀₀ of 0.5 to 1.0 is obtained.

Chill the culture on ice. Centrifuge the cell suspension at 1000 g for 30 minutes at 4ºC.

Discard the supernatant. Resuspend the cells in 1 ml of 20 mM CaCl₂ (ice cold). Dispense 100 mL aliquots into pre-chilled 1.5 ml tubes.

Add about 1 µg of the desired plasmid DNA (pure or crude) to the cells in each tube.

This protocol is designed for potato stem internodes on ZIG medium, but might work for leaf pieces as well.

Plant Material

Cut stem internode explants from 5-6-week-old in vitro plants (or leaf explants from 3-4-week-old in vitro plants) grown on 15 ml PROP in 25 x 150 mm test tubes.

Day 1

Streak out desired Agrobacterium strain on YEP + antibiotic to obtain a single colony. Grow at 28 C for 2 days. Alternatively, skip this step and use frozen stock for next step.

Day 3

Use a single Agrobacterium colony to inoculate 20 ml YEP + antibiotic in 250-ml flask. Grow overnight at 28 C with vigorous shaking (~200 rpm). Some Agrobacterium strains require an additional day to reach sufficient density.

Principle

Micropropagation is especially useful for include propagation of high value plants that grow slowly (bulbs, ferns, orchids, palms, etc), new cultivars having limited stock and high market demand (new cultivars produced via conventional breeding or genetic engineering), cultivars that are difficult to root by conventional techniques, and endangered plant species. In addition, virus elimination is possible via culture initiation from meristem explants. Also, micropropagation provides a source of sterile tissues that can be used for other tissue culture protocols, including genetic transformation.

Micropropagation is possible using multiple types of cultures; however, shoot cultures are typically employed, in which case five stages to the micropropagation process are typically recognized as outlined below. Nodal explants of rose are used for culture initiation. Three media, differing in hormone content, is employed and the explants should be evaluated for differences in axillary shoot growth and proliferation.

Principle

Orchid seeds are produced in large numbers but are exceedingly small, hence are also called “dust seeds” may be as small as 0.01 by 0.05 mm in dimension and weigh as little as 10-30 ug. Seedling development is generally a slow process in orchids, with the seedlings passing through an initial stage, termed the protocorm stage, prior to the production of distinct vegetative organs (leaves, roots, etc). The protocorms of terrestrial orchids are subterranean and entirely heterotrophic, with all of the nutritional needs of the orchid being supplied by the mycorrhizae (the symbiotic fungus). The length of the protocorm stage is variable, however, in many cases, months, or even years, elapse before leaves and roots are produced.

The combination of a small seed size and fungal symbiont requirement largely precludes the propagation via conventional seed propagation protocols. Accordingly, most of the early research on the development of orchid seed germination protocols focused on in vitro germination with the seeds and fungal symbiont being sown on the same petri plate. Successful protocols were developed for several species; however, the process was complicated by the diversity of fungal symbionts required (across orchid taxa) and the difficulty in isolating and culturing these fungi.

In the 1920’s the process of orchid seed propagation was revolutionized by Lewis Knudson with the demonstration that nutrient media that could be used as a substitute for fungal infection. The isolation and culture of multiple fungal strains was no longer required, and improvements in germination frequency, germination speed, and seedling vigor were generally observed. To date, successful asymbiotic germination techniques, termed ‘flasking’, have been developed for many epiphytic orchids, with several species being saved from extinction via the application of these techniques.

Media Preparation

Before mixing medium, determine the quantity of each medium and the amount of plant growth regulator needed for each experiment. Make slightly more medium than is required. A media check off list is helpful in making medium to keep track of ingredients.

Remove media stock solutions and plant growth regulator stock solutions to be used from the refrigerator. Make up fresh stock solutions if adequate quantities are not available or if solutions have precipitated.

Plant growth regulators stock solutions are prepared biweekly and stored at 4º C in the dark. Cytokinins (thidiazuron and BA) are dissolved in approximately KOH prior the addition of deionized water. Gibberellins and auxins (2,4-D, IBA and NAA) are dissolved in 100% ethanol prior the addition of water.

Fill the volumetric flasks upto ½ to ¾ with deionized water. Add proper a of stock solution and plant growth regulators and mark each item on the check off list. Add 30 g/l sucrose, stir until dissolved. Make up the volume of the flask with deionized water and later por into a larger beaker.